Human Patched genes and proteins, and uses related thereto

ABSTRACT

The present invention relates to the discovery of a new member of the hedgehog receptor family, referred to herein as human ptc-2 (for patched-2 protein). The human ptc-2 polypeptides of the present invention include polypeptides which bind the products of the hedgehog gene family. Hedgehog family members are known for their broad involvement in the formation and maintenance of ordered spatial arrangements of differentiated tissues in vertebrates, both adult and embryonic, and can be used to generate and/or maintain an array of different vertebrate tissue both in vitro and in vivo.

BACKGROUND OF THE INVENTION

[0001] Pattern formation is the activity by which embryonic cells formordered spatial arrangements of differentiated tissues. The physicalcomplexity of higher organisms arises during embryogenesis through theinterplay of cell-intrinsic lineage and cell-extrinsic signaling.Inductive interactions are essential to embryonic patterning invertebrate development from the earliest establishment of the body plan,to the patterning of the organ systems, to the generation of diversecell types during tissue differentiation (Davidson, E., (1990)Development 108: 365-389; Gurdon, J. B., (1992) Cell 68: 185-199;Jessell, T. M. et al.,.(1992) Cell 68: 257-270). The effects ofdevelopmental cell interactions are varied. Typically, responding cellsare diverted from one route of cell differentiation to another byinducing cells that differ from both the uninduced and induced states ofthe responding cells (inductions). Sometimes cells induce theirneighbors to differentiate like themselves (homoiogenetic induction); inother cases a cell inhibits its neighbors from differentiating likeitself. Cell interactions in early development may be sequential, suchthat an initial induction between two cell types leads to a progressiveamplification of diversity. Moreover, inductive interactions occur notonly in embryos, but in adult cells as well, and can act to establishand maintain morphogenetic patterns as well as induce differentiation(J. B. Gurdon (1992) Cell 68:185-199).

[0002] The origin of the nervous system in all vertebrates can be tracedto the end of gastrulation. At this time, the ectoderm in the dorsalside of the embryo changes its fate from epidermal to neural. The newlyformed neuroectoderm thickens to form a flattened structure called theneural plate which is characterized, in some vertebrates, by a centralgroove (neural groove) and thickened lateral edges (neural folds). Atits early stages of differentiation, the neural plate already exhibitssigns of regional differentiation along its anterior posterior (A-P) andmediolateral axis (M-L). The neural folds eventually fuse at the dorsalmidline to form the neural tube which will differentiate into brain atits anterior end and spinal cord at its posterior end. Closure of theneural tube creates dorsal/ventral differences by virtue of previousmediolateral differentiation. Thus, at the end of neurulation, theneural tube has a clear anterior-posterior (A-P), dorsal ventral (D-V)and mediolateral (M-L) polarities (see, for example, Principles inNeural Science (3rd), eds. Kandel, Schwartz and Jessell, ElsevierScience Publishing Company: NY, 1991; and Developmental Biology (3rd),ed. S. F. Gilbert, Sinauer Associates: Sunderland Mass., 1991).Inductive interactions that define the fate of cells within the neuraltube establish the initial pattern of the embryonic vertebrate nervoussystem. In the spinal cord, the identify of cell types is controlled, inpart, by signals from two midline cell groups, the notochord and floorplate, that induce neural plate cells to differentiate into floor plate,motor neurons, and other ventral neuronal types (van Straaten et al.(1988) Anat. Embryol. 177:317-324; Placzek et al. (1993) Development117:205-218; Yamada et al. (1991) Cell 64:035-647; and Hatta et al.(1991) Nature 350:339-341). In addition, signals from the floor plateare responsible for the orientation and direction of commissural neuronoutgrowth (Placzek, M. et al., (1990) Development 110: 19-30). Besidespatterning the neural tube, the notochord and floorplate are alsoresponsible for producing signals which control the patterning of thesomites by inhibiting differentiation of dorsal somite derivatives inthe ventral regions (Brand-Saberi, B. et al., (1993) Anat. Embryol. 188:239-245; Porquie, O et al., (1993) Proc. Natl Acad. Sci. USA 90:5242-5246).

[0003] Another important signaling center exists in the posteriormesenchyme of developing limb buds, called the Zone of PolarizingActivity, or “ZPA”. When tissue from the posterior region of the limbbud is grafted to the anterior border of a second limb bud, theresultant limb will develop with additional digits in a mirror-imagesequence along the anteroposterior axis (Saunders and Gasseling, (1968)Epithelial-Mesenchymal Interaction, pp. 78-97). This finding has led tothe model that the ZPA is responsible for normal anteroposteriorpatterning in the limb. The ZPA has been hypothesized to function byreleasing a signal, termed a “morphogen”, which forms a gradient acrossthe early embryonic bud. According to this model, the fate of cells atdifferent distances from the ZPA is determined by the localconcentration of the morphogen, with specific thresholds of themorphogen inducing successive structures (Wolpert, (1969) Theor. Biol.25:1-47). This is supported by the finding that the extent of digitduplication is proportional to the number of implanted ZPA cells(Tickle, (1981) Nature 254:199-202).

[0004] Although the existence of inductive signals in the ZPA has beenknown for years, the molecular identities of these signals are only nowbeginning to be elucidated. An important step forward has been thediscovery that the secreted protein Sonic hedgehog (Shh) is produced inseveral tissues with organizing properties, including notochord, floorplate and ZPA (Echelard et al. (1993), Cell 75: 1417-1430; Bitgood, M.J. and A. P. McMahon (1995) Dev. Biol. 172:126-38). Misexpressing Shhmimics the inductive effects on ectopic notochord in the neural tube andsomites (Echelard et al. (1993) supra) and also mimics ZPA function inthe limb bud (Riddle et al. (1993) Cell 75:1401-16; Chang et al. (1994)Development 120: 3339-53).

[0005] The vertebrate family of hedgehog genes includes at least fourmembers, e.g., paralogs of the single drosophila hedgehog gene.Exemplary hedgehog genes and proteins are described in PCT publicationsWO 95/18856 and WO 96/17924. Three of these members, herein referred toas Desert hedgehog (Dhh), Sonic hedgehog (Shh) and Indian hedgehog(Ihh), apparently exist in all vertebrates, including fish, birds, andmammals. A fourth member, herein referred to as tiggie-winkle hedgehog(Thh), appears specific to fish. Desert hedgehog (Dhh) is expressedprincipally in the testes, both in mouse embryonic development and inthe adult rodent and human; Indian hedgehog (Ihh) is involved in bonedevelopment during embryogenesis and in bone formation in the adult;and, Shh, which as described above, is primarily involved in morphogenicand neuroinductive activities. Given the critical inductive roles ofhedgehog polypeptides in the development and maintenance of vertebrateorgans, the identification of hedgehog interacting proteins is ofparamount significance in both clinical and research contexts.

SUMMARY OF THE INVENTION

[0006] The present invention relates to the discovery of a new member ofthe hedgehog receptor family, referred to herein as human ptc-2 (forpatched-2 protein). The human ptc-2 polypeptides of the presentinvention include polypeptides which bind the products of the hedgehoggene family. Hedgehog family members are known for their broadinvolvement in the formation and maintenance of ordered spatialarrangements of differentiated tissues in vertebrates, both adult andembryonic, and can be used to generate and/or maintain an array ofdifferent vertebrate tissue both in vitro and in vivo.

[0007] In general, the invention features isolated ptc-2 polypeptides,preferably substantially pure preparations of the subject ptc-2polypeptides. The invention also provides recombinantly produced humanptc-2 polypeptides.

[0008] In one embodiment, the polypeptide is identical with orhomologous to the ptc-2 polypeptide represented in SEQ ID No: 2.

[0009] The ptc-2 polypeptide can comprise a full length protein, or itmay include only a hedgehog-binding portion thereof, or it may be ofarbitrary sizes, e.g., at least 5, 10, 25, 50, 100, 150 or 200 aminoacids in length. In preferred embodiments, the ptc-2 polypeptideincludes a sufficient portion of the extracellular ligand binding domainto be able to specifically bind to a hedgehog ligand. Truncated forms ofthe protein include, but are not limited to, soluble ligand bindingdomain fragments.

[0010] In yet another embodiment, the invention features nucleic acidsencoding ptc-2 polypeptides, which have the ability to modulate, e.g.,either mimic or antagonize, at least a portion of the activity of awild-type ptc-2 polypeptide. Exemplary ptc-2-encoding nucleic acidsequences are represented by SEQ ID No: 1. In another embodiment, thenucleic acids of the present invention include coding sequences whichhybridize under stringent conditions with all or a portion of the codingsequences designated in SEQ ID No: 1.

[0011] Furthermore, in certain preferred embodiments, the subject ptc-2nucleic acids will include a transcriptional regulatory sequence, e.g.at least one of a transcriptional promoter or transcriptional enhancersequence, which regulatory sequence is operably linked to the ptc-2 genesequences. Such regulatory sequences can be used in to render the ptc-2gene sequences suitable for use as an expression vector. Thetranscriptional regulatory sequence can be from a ptc-2 gene, or from aheterologous gene.

[0012] This invention also contemplates the cells transfected with saidexpression vector whether prokaryotic or eukaryotic and a method forproducing ptc-2 proteins by employing said expression vectors.

[0013] In still other embodiments, the subject invention provides a geneactivation construct, wherein the gene activation construct is deignedto recombine with a genomic ptc-2 gene in a cell to provide, e.g., byheterologous recombination, a heterologous transcriptional regulatorysequence operatively linked to a coding sequence of a genomic ptc-2gene. Cells having genomic ptc-2 genes modified by gene activationconstructs are also specifically contemplated.

[0014] A still further aspect of the present invention featuresantibodies and antibody preparations specifically reactive with anepitope of the ptc-2 immunogen.

[0015] The invention also provides a probe/primer comprising asubstantially purified oligonucleotide, wherein the oligonucleotidecomprises a region of nucleotide sequence which hybridizes understringent conditions to at least 12 consecutive nucleotides of sense orantisense sequences of any one or more of SEQ ID No: 1, or naturallyoccurring mutants thereof. In preferred embodiments, the probe/primerfurther includes a label group attached thereto and able to be detected.The label group can be selected, e.g., from a group consisting ofradioisotopes, fluorescent compounds, enzymes, and enzyme co-factors.Probes of the invention can be used as a part of a diagnostic test kitfor identifying dysfunctions associated with mis-expression of a ptc-2protein, such as for detecting in a sample of cells isolated from apatient, a level of a nucleic acid encoding a ptc-2 protein; e.g.measuring a ptc-2 mRNA level in a cell, or determining whether a genomicptc-2 gene has been mutated or deleted. These so-called “probes/primers”of the invention can also be used as a part of “antisense” therapy whichrefers to administration or in situ generation of oligonucleotide probesor their derivatives which specifically hybridize (e.g. bind) undercellular conditions, with the cellular mRNA and/or genomic DNA encodingone or more of the subject ptc-2 proteins so as to inhibit expression ofthat protein, e.g. by inhibiting transcription and/or translation.Preferably, the oligonucleotide is at least 12 nucleotides in length,though primers of 25, 40, 50, or 75 nucleotides in length are alsocontemplated.

[0016] In yet another aspect, the invention provides an assay forscreening test compounds for inhibitors, or alternatively, potentiators,of an interaction between a hedgehog protein and a ptc-2 polypeptidereceptor. An exemplary method includes the steps of (a) forming areaction mixture including: (i) a hedgehog polypeptide, (ii) a ptc-2polypeptide, and (iii) a test compound; and (b) detecting interaction ofthe hedgehog and ptc-2 polypeptides. A statistically significant change(potentiation or inhibition) in the interaction of the hedgehog andptc-2 polypeptides in the presence of the test compound, relative to theinteraction in the absence of the test compound, indicates a potentialagonist (mimetic or potentiator) or antagonist (inhibitor) of hedgehogbioactivity for the test compound. The reaction mixture—can be acell-free protein preparation, e.g., a reconstituted protein mixture ora cell lysate, or it can be a recombinant cell including a heterologousnucleic acid recombinantly expressing the ptc-2 polypeptide.

[0017] In preferred embodiments, the step of detecting interaction ofthe hedgehog and ptc-2 polypeptides is a competitive binding assay. Inother preferred embodiments, the step of detecting interaction of thehedgehog and ptc-2 polypeptides involves detecting, in a cell-basedassay, change(s) in the level of an intracellular second messengerresponsive to signaling mediated by the ptc-2 polypeptide. In stillanother preferred embodiment, the step of detecting interaction of thehedgehog and ptc-2 polypeptides comprises detecting, in a cell-basedassay, change(s) in the level of expression of a gene controlled by atranscriptional regulatory sequence responsive to signaling by the ptc-2polypeptide.

[0018] In preferred embodiments, the steps of the assay are repeated fora variegated library of at least 100 different test compounds, morepreferably at least 10^(3, 10) ⁴ or 10⁵ different test compounds. Thetest compound can be, e.g., a peptide, a nucleic acid, a carbohydrate, asmall organic molecule, or natural product extract (or fractionthereof).

[0019] The present invention further contemplates the pharmaceuticalformulation of one or more agents identified in such drug screeningassays.

[0020] In other embodiments, the present invention provides a molecule,preferably a small organic molecule, which binds to ptc-2 and eithermimics or antagonizes hedgehog-induced signaling in cells expressingptc-2.

[0021] Yet another aspect of the present invention concerns a method formodulating one or more of growth, differentiation, or survival of a cellby modulating ptc-2 bioactivity, e.g., by potentiating or disruptingcertain protein-protein interactions. In general, whether carried out invivo, in vitro, or in situ, the method comprises treating the cell withan effective amount of a ptc-2 therapeutic so as to alter, relative tothe cell in the absence of treatment, at least one of (i) rate ofgrowth, (ii) differentiation, or (iii) survival of the cell.Accordingly, the method can be carried out with ptc-2 therapeutics suchas peptide and peptidomimetics or other molecules identified in theabove-referenced drug screens which agonize or antagonize the effects ofsignaling from a ptc-2 protein or ligand binding of a ptc-2 protein,e.g., a hedgehog protein. Other ptc-2 therapeutics include antisenseconstructs for inhibiting expression of ptc-2 proteins, dominantnegative mutants of ptc-2 proteins which competitively inhibit ligandinteractions upstream and signal transduction downstream of thewild-type ptc-2 protein, and gene therapy constructs including geneactivation constructs.

[0022] In one embodiment, the subject method of modulating ptc-2bioactivity can be used in the treatment of testicular cells, so as tomodulate spermatogenesis. In another embodiment, the subject method isused to modulate osteogenesis, comprising the treatment of osteogeniccells with an agent that modulates ptc-2 bioactivity. Likewise, wherethe treated cell is a chondrogenic cell, the present method is used tomodulate chondrogenesis. In still, another embodiment, the subjectmethod can be used to modulate the differentiation of a neuronal cell,to maintain a neuronal cell in a differentiated state, and/or to enhancethe survival of a neuronal cell, e.g., to prevent apoptosis or otherforms of cell death. For instance the present method can be used toaffect the differentiation of neuronal cells such as motor neurons,cholinergic neurons, dopaminergic neurons, serotonergic neurons, andpeptidergic neurons.

[0023] Another aspect of the present invention provides a method ofdetermining if a subject, e.g. an animal patient, is at risk for adisorder characterized by unwanted cell proliferation or aberrantcontrol of differentiation or apoptosis. The method includes detecting,in a tissue of the subject, the presence or absence of a genetic lesioncharacterized by at least one of (i) a mutation of a gene encoding aptc-2 protein; or (ii) the mis-expression of a ptc-2 gene. In preferredembodiments, detecting the genetic lesion includes ascertaining theexistence of at least one of: a deletion of one or more nucleotides froma ptc-2 gene; an addition of one or more nucleotides to the gene, asubstitution of one or more nucleotides of the gene, a gross chromosomalrearrangement of the gene; an alteration in the level of a messenger RNAtranscript of the gene; the presence of a non-wild type splicing patternof a messenger RNA transcript of the gene; a non-wild type level of theprotein; and/or an aberrant level of soluble ptc-2 protein.

[0024] For example, detecting the genetic lesion can include (i)providing a probe/primer including an oligonucleotide containing aregion of nucleotide sequence which hybridizes to a sense or antisensesequence of a ptc-2 gene or naturally occurring mutants thereof, or 5′or 3′ flanking sequences naturally associated with the ptc-2 gene; (ii)exposing the probe/primer to nucleic acid of the tissue; and (iii)detecting, by hybridization of the probe/primer to the nucleic acid, thepresence or absence of the genetic lesion; e.g. wherein detecting thelesion comprises utilizing the probe/primer to determine the nucleotidesequence of the ptc-2 gene and, optionally, of the flanking nucleic acidsequences. For instance, the probe/primer can be employed in apolymerase chain reaction (PCR) or in a ligation chain reaction (LCR).In alternate embodiments, the level of a ptc-2 protein is detected in animmunoassay using an antibody which is specifically immunoreactive withthe ptc-2 protein.

[0025] The practice of the present invention will employ, unlessotherwise indicated, conventional techniques of cell biology, cellculture, molecular biology, transgenic biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See, for example,Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No: 4,683,195; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

[0026] Other features and advantages of the invention will be apparentfrom the following detailed description, and from the claims.

DETAILED DESCRIPTION OF THE INVENTION

[0027] Of particular importance in the development and maintenance oftissue in vertebrate animals is a type of extracellular communicationcalled induction, which occurs between neighboring cell layers andtissues. In inductive interactions, chemical signals secreted by onecell population influence the developmental fate of a second cellpopulation. Typically, cells responding to the inductive signals arediverted from one cell fate to another, neither of which is the same asthe fate of the signaling cells.

[0028] Inductive signals are key regulatory proteins that function invertebrate pattern formation, and are present in important signalingcenters known to operate embryonically, for example, to define theorganization of the vertebrate embryo. For example, these signalingstructures include the notochord, a transient structure which initiatesthe formation of the nervous system and helps to define the differenttypes of neurons within it. The notochord also regulates mesodermalpatterning along the body axis. Another distinct group of cells havingapparent signaling activity is the floorplate of the neural tube (theprecursor of the spinal cord and brain) which also signals thedifferentiation of different nerve cell types. It is also generallybelieved that the region of mesoderm at the bottom of the buds whichform the limbs (called the Zone of Polarizing Activity or ZPA) operatesas a signaling center by secreting a morphogen which ultimately producesthe correct patterning of the developing limbs.

[0029] The regulation of hedgehog protein signaling is an importantmechanism for developmental control. The present invention concerns thediscovery of a new member of the hedgehog receptor family, referred toherein as “patched-2” or “ptc-2”.

[0030] The sequence of an exemplary human ptc-2 gene (cf, SEQ ID No. 1)indicates it encodes a serpentine membrane protein. The ptc-2 proteins,through their ability to bind to hedgehog proteins, are apparentlycapable of modulating hedgehog signaling. The ptc-2 proteins canfunction as a hedgehog receptor (or subunit thereof). Thus, the ptc-2polypeptides of the present invention may affect a number ofhedgehog-mediated biological activities including: an ability tomodulate proliferation, survival and/or differentiation ofmesodermally-derived tissue, such as tissue derived from dorsalmesoderm, cartilage and tissue involved in spermatogenesis; the abilityto modulate proliferation, survival and/or differentiation ofectodermally-derived tissue, such as tissue derived from the epidermis,neural tube, neural crest, or head mesenchyme; the ability to modulateproliferation, survival and/or differentiation of endodermally-derivedtissue, such as tissue derived from the primitive gut.

[0031] Accordingly, certain aspects of the present invention relate tonucleic acids encoding ptc-2 polypeptides, the ptc-2 polypeptidesthemselves (including various fragments), antibodies immunoreactive withptc-2 proteins, and preparations of such compositions. Moreover, thepresent invention provides diagnostic and therapeutic assays andreagents for detecting and treating disorders involving, for example,aberrant expression (or loss thereof) of ptc-2, ptc-2 ligands(particularly hedgehog proteins), or signal transducers thereof.

[0032] In addition, drug discovery assays are provided for identifyingagents which can modulate the biological function of ptc-2 proteins,such as by altering the binding of ptc-2 molecules to hedgehog proteinsor other extracellular/matrix factors, or the ability of the bound ptc-2protein to transduce hedgehog signals. Such agents can be usefultherapeutically to alter the growth, maintenance and/or differentiationof a tissue, particularly a mesodermally-derived tissue, such cartilage,tissue involved in spermatogenesis and tissue derived from dorsalmesoderm; ectodermally-derived tissue, such as tissue derived from theepidermis, neural tube, neural crest, or head mesenchyme;endodermally-derived tissue, such as tissue derived from the primitivegut. Other aspects of the invention are described below or will beapparent to those skilled in the art in light of the present disclosure.

[0033] For convenience, certain terms employed in the specification andappended claims are collected here.

[0034] The term “human ptc-2” polypeptide refers to polypeptidescharacterized at least in part by being identical or sharing a degree ofsequence homology with all or a portion of the a ptc-2 polypeptiderepresented in SEQ ID No: 2.

[0035] A “glycosylated” ptc-2 polypeptide is an ptc-2 polypeptide havinga covalent linkage with a glycosyl group (e.g. a derivatized with acarbohydrate). For instance, the ptc-2 protein can be glycosylated on anexisting residue, or can be mutated to preclude carbohydrate attachment,or can be mutated to provide new glycosylation sites, such as forN-linked or O-linked glycosylation.

[0036] As used herein, the term “vertebrate hedgehog protein” refers tovertebrate inter-cellular signaling molecules related to the Drosophilahedgehog protein. Three of the vertebrate hedgehog proteins, Deserthedgehog (Dhh), Sonic hedgehog (Shh) and Indian hedgehog (Ihh),apparently exist in all vertebrates, including amphibians, fish, birds,and mammals. Other members of this family, such as Banded hedgehog,Cephalic hedgehog, tiggy-winkle hedgehog, and echidna hedgehog have beenso far identified in fish and/or amphibians. Exemplary hedgehogpolypeptides are described in PCT applications W096/17924, W096/16668,W095/18856.

[0037] As used herein, the term “nucleic acid” refers to polynucleotidessuch as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleicacid (RNA). The term should also be understood to include, asequivalents, analogs of either RNA or DNA made from nucleotide analogs,and, as applicable to the embodiment being described, single (sense orantisense) and double-stranded polynucleotides.

[0038] As used herein, the term “gene” or “recombinant gene” refers to anucleic acid comprising an open reading frame encoding a ptc-2polypeptide, including both exon and (optionally) intron sequences. A“recombinant gene” refers to nucleic acid encoding a ptc-2 polypeptideand comprising ptc-2-encoding exon sequences, though it may optionallyinclude intron sequences which are derived from, for example, achromosomal ptc-2 gene or from an unrelated chromosomal gene. Exemplaryrecombinant genes encoding the subject ptc-2 polypeptide are representedin the appended Sequence Listing. The term “intron” refers to a DNAsequence present in a given ptc-2 gene which is not translated intoprotein and is generally found between exons.

[0039] As used herein, the term “transfection” means the introduction ofa nucleic acid, e.g., an expression vector, into a recipient cell bynucleic acid-mediated gene transfer. “Transformation”, as used herein,refers to a process in which a cell's genotype is changed as a result ofthe cellular uptake of exogenous DNA or RNA, and, for example, thetransformed cell expresses a recombinant form of a ptc-2 polypeptide or,where anti-sense expression occurs from the transferred gene, theexpression of a naturally-occurring form of the ptc-2 protein isdisrupted.

[0040] As used herein, the term “specifically hybridizes” refers to theability of a nucleic acid probe/primer of the invention to hybridize toat least 15 consecutive nucleotides of a ptc-2 gene, such as the ptc-2sequence designated in SEQ ID No: 1, or a sequence complementarythereto, or naturally occurring mutants thereof, such that it has lessthan 15%, preferably less than 10%, and more preferably less than 5%background hybridization to a cellular nucleic acid (e.g., mRNA orgenomic DNA) encoding a protein other than a ptc-2 protein, as definedherein.

[0041] An “effective amount” of a hedgehog polypeptide, or a bioactivefragment thereof, with respect to the subject method of treatment,refers to an amount of agonist or antagonist in a preparation which,when applied as part of a desired dosage regimen, provides modulation ofgrowth, differentiation or survival of cells, e.g., modulation ofspermatogenesis, neuronal differentiation, or skeletogenesis, e.g.,osteogenesis, chondrogenesis, or limb patterning.

[0042] As used herein, “phenotype” refers to the entire physical,biochemical, and physiological makeup of a cell, e.g., having any onetrait or any group of traits.

[0043] The terms “induction” or “induce”, as relating to the biologicalactivity of a hedgehog protein, refers generally to the process or actof causing to occur a specific effect on the phenotype of cell. Sucheffect can be in the form of causing a change in the phenotype, e.g.,differentiation to another cell phenotype, or can be in the form ofmaintaining the cell in a particular cell, e.g., preventingdedifferentation or promoting survival of a cell.

[0044] A “patient” or “subject” to be treated can mean either a human ornon-human animal.

[0045] As used herein, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of preferred vector is an episome, i.e., a nucleicacid capable of extra-chromosomal replication. Preferred vectors arethose capable of autonomous replication and/expression of nucleic acidsto which they are linked. Vectors capable of directing the expression ofgenes to which they are operatively linked are referred to herein as“expression vectors”. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of “plasmids” whichrefer generally to circular double stranded DNA loops which, in theirvector form are not bound to the chromosome. In the presentspecification, “plasmid” and “vector” are used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors whichserve equivalent functions and which become known in the artsubsequently hereto.

[0046] “Transcriptional regulatory sequence” is a generic term usedthroughout the specification to refer to DNA sequences, such asinitiation signals, enhancers, and promoters, which induce or controltranscription of protein coding sequences with which they are operablylinked. In preferred embodiments, transcription of a recombinant ptc-2gene is under the control of a promoter sequence (or othertranscriptional regulatory sequence) which controls the expression ofthe recombinant gene in a cell-type in which expression is intended. Itwill also be understood that the recombinant gene can be under thecontrol of transcriptional regulatory sequences which are the same orwhich are different from those sequences which control transcription ofthe naturally-occurring forms of ptc-2 genes.

[0047] As used herein, the term “tissue-specific promoter” means a DNAsequence that serves as a promoter, i.e., regulates expression of aselected DNA sequence operably linked to the promoter, and which effectsexpression of the selected DNA sequence in specific cells of a tissue,such as cells of neuronal or hematopoietic origin. The term also coversso-called “leaky” promoters, which regulate expression of a selected DNAprimarily in one tissue, but can cause at least low level expression inother tissues as well.

[0048] As used herein, the term “target tissue” refers to connectivetissue, cartilage, bone tissue or limb tissue, which is either presentin an animal, e.g., a mammal, e.g., a human or is present in vitroculture, e.g., a cell culture.

[0049] As is well known, genes for a particular polypeptide may exist insingle or multiple copies within the genome of an individual. Suchduplicate genes may be identical or may have certain modifications,including nucleotide substitutions, additions or deletions, which allstill code for polypeptides having substantially the same activity. Theterm “DNA sequence encoding a ptc-2 polypeptide” may thus refer to oneor more genes within a particular individual. Moreover, certaindifferences in nucleotide sequences may exist between individuals of thesame species, which are called alleles. Such allelic differences may ormay not result in differences in amino acid sequence of the encodedpolypeptide yet still encode a protein with the same biologicalactivity.

[0050] “Homology” and “identity” each refer to sequence similaritybetween two polypeptide sequences, with identity being a more strictcomparison. Homology and identity can each be determined by comparing aposition in each sequence which may be aligned for purposes ofcomparison. When a position in the compared sequence is occupied by thesame amino acid residue, then the polypeptides can be referred to asidentical at that position; when the equivalent site is occupied by thesame amino acid (e.g., identical) or a similar amino acid (e.g., similarin steric and/or electronic nature), then the molecules can be referredto as homologous at that position. A percentage of homology or identitybetween sequences is a function of the number of matching or homologouspositions shared by the sequences. An “unrelated” or “non-homologous”sequence shares less than 40 percent identity, though preferably lessthan 25 percent identity, with a ptc-2 sequence of the presentinvention.

[0051] “Cells,” “host cells” or “recombinant host cells” are terms usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

[0052] A “chimeric protein” or “fusion protein” is a fusion of a firstamino acid sequence encoding a ptc-2 polypeptide with a second aminoacid sequence defining a domain (e.g. polypeptide portion) foreign toand not substantially homologous with any domain of a ptc-2 protein. Achimeric protein may present a foreign domain which is found (albeit ina different protein) in an organism which also expresses the firstprotein, or it may be an “interspecies”, “intergenic”, etc. fusion ofprotein structures expressed by different kinds of organisms. Ingeneral, a fusion protein can be represented by the general formulaX-ptc-2-Y, wherein ptc-2 represents a portion of the fusion proteinwhich is derived from a ptc-2 protein, and X and Y are, independently,absent or represent amino acid sequences which are not related to aptc-2 sequences in an organism.

[0053] As used herein, a “reporter gene construct” is a nucleic acidthat includes a “reporter gene” operatively linked to a transcriptionalregulatory sequences. Transcription of the reporter gene is controlledby these sequences. The activity of at least one or more of thesecontrol sequences is directly or indirectly regulated by a signaltransduction pathway involving a phospholipase, e.g., is directly orindirectly regulated by a second messenger produced by the phospholipaseactivity. The transcriptional regulatory sequences can include apromoter and other regulatory regions, such as enhancer sequences, thatmodulate the activity of the promoter, or regulatory sequences thatmodulate the activity or efficiency of the RNA polymerase thatrecognizes the promoter, or regulatory sequences that are recognized byeffector molecules, including those that are specifically induced uponactivation of a phospholipase. For example, modulation of the activityof the promoter may be effected by altering the RNA polymerase bindingto the promoter region, or, alternatively, by interfering withinitiation of transcription or elongation of the mRNA. Such sequencesare herein collectively referred to as transcriptional regulatoryelements or sequences. In addition, the construct may include sequencesof nucleotides that alter the stability or rate of translation of theresulting mRNA in response to second messages, thereby altering theamount of reporter gene product.

[0054] The term “isolated” as also used herein with respect to nucleicacids, such as DNA or RNA, refers to molecules separated from otherDNAs, or RNAs, respectively, that are present in the natural source ofthe macromolecule. For example, an isolated nucleic acid encoding aptc-2 polypeptide preferably includes no more than 10 kilobases (kb) ofnucleic acid sequence which naturally immediately flanks the ptc-2 genein genomic DNA, more preferably no more than 5 kb of such naturallyoccurring flanking sequences, and most preferably less than 1.5 kb ofsuch naturally occurring flanking sequence. The term isolated as usedherein also refers to a nucleic acid or peptide that is substantiallyfree of cellular material, or culture medium when produced byrecombinant DNA techniques, or chemical precursors or other chemicalswhen chemically synthesized. Moreover, an “isolated nucleic acid” ismeant to include nucleic acid fragments which are not naturallyoccurring as fragments and would not be found in the natural state.

[0055] As described below, one aspect of the invention pertains toisolated nucleic acids comprising nucleotide sequences encoding ptc-2polypeptides, and/or equivalents of such nucleic acids. The term nucleicacid as used herein is intended to include fragments as equivalents. Theterm equivalent is understood to include nucleotide sequences encodingfunctionally equivalent ptc-2 polypeptides or functionally equivalentpeptides having an activity of a ptc-2 protein such as described herein.Equivalent nucleotide sequences will include sequences that differ byone or more nucleotide substitutions, additions or deletions, such asallelic variants; and will, therefore, include sequences that differfrom the nucleotide sequence of the human ptc-2 coding sequence of SEQID No: 1 due to the degeneracy of the genetic code. Equivalents willalso include nucleotide sequences that hybridize under stringentconditions (i.e., equivalent to about 20-27° C. below the meltingtemperature (T_(m)) of the DNA duplex formed in about 1M salt) to thenucleotide sequences represented in SEQ ID No: 1.

[0056] Moreover, it will be generally appreciated that, under certaincircumstances, it may be advantageous to provide homologs of a ptc-2polypeptide which function in a limited capacity as one of either anagonist (e.g., mimics or potentiates a bioactivity of the wild-typeptc-2 protein) or an antagonist (e.g., inhibits a bioactivity of thewild-type ptc-2 protein), in order to promote or inhibit only a subsetof the biological activities of the naturally-occurring form of theprotein. Thus, specific biological effects can be elicited by treatmentwith a homolog of limited function. For example, truncated forms of thepatched-2 protein, e.g., soluble fragments of an extracellular domain,can be provided to competitively inhibit ligand (hedgehog) binding tothe wild-type ptc-2 protein.

[0057] Homologs of the subject ptc-2 protein can be generated bymutagenesis, such as by discrete point mutation(s), or by truncation.For instance, mutation can give rise to homologs which retainsubstantially the same, or merely a subset, of the biological activityof the ptc-2 polypeptide from which it was derived. Alternatively,antagonistic forms of the protein can be generated which are able toinhibit the function of the naturally occurring form of the protein,such as by competitively binding to hedgehog proteins and competing withwild-type ptc-2, or binding to other patched-2 proteins (such assubunits of a hedgehog receptor) to form unresponsive hedgehog receptorcomplexes. Thus, the ptc-2 protein and homologs thereof provided by thesubject invention may be either positive or negative regulators of cellgrowth, death and/or differentiation.

[0058] In general, polypeptides referred to herein as having an activityof a ptc-2 protein (e.g., are “bioactive”) are defined as polypeptideswhich include an amino acid sequence corresponding (e.g., identical orhomologous) to all or a portion of the amino acid sequences of the ptc-2protein shown in SEQ ID No: 2, and which agonize or antagonize all or aportion of the biological/biochemical activities of a naturallyoccurring ptc-2 protein. Examples of such biological activity includesthe ability to bind with high affinity hedgehog proteins. Thebioactivity of certain embodiments of the subject ptc-2 polypeptides canbe characterized in terms of an ability to promote differentiationand/or maintenance of cells and tissue from mesodermally-derived tissue,such as tissue derived from dorsal mesoderm; ectodermally-origin, suchas tissue derived from the neural tube, neural crest, or headmesenchyme; or endodermally-derived tissue, such as tissue derived fromthe primitive gut.

[0059] Other biological activities of the subject ptc-2 proteins aredescribed herein or will be reasonably apparent to those skilled in theart. According to the present invention, a polypeptide has biologicalactivity if it is a specific agonist or antagonist of anaturally-occurring form of a ptc-2 protein.

[0060] Preferred nucleic acids encode a ptc-2 polypeptide comprising anamino acid sequence at least 80%, 85% or 90% homologous, more preferablyat least 93% homologous and most preferably at least 95% homologous withan amino acid sequence of a naturally occurring ptc-2 protein, e.g.,such as represented in SEQ ID No: 2. Nucleic acids which encodepolypeptides at least about 98-99% homology with an amino acid sequencerepresented in SEQ ID No: 2 are of course also within the scope of theinvention, as are nucleic acids identical in sequence with theenumerated ptc-2 sequence of the Sequence listing. In one embodiment,the nucleic acid is a cDNA encoding a polypeptide having at least oneactivity of the subject ptc-2 polypeptide.

[0061] Another aspect of the invention provides a nucleic acid whichhybridizes under high or low stringency conditions to the nucleic acidsrepresented by SEQ ID No: 1. Appropriate stringency conditions whichpromote DNA hybridization, for example, 6.0×sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C.,are known to those skilled in the art or can be found in CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6. For example, the salt concentration in the wash step can beselected from a low stringency of about 2.0×SSC at 50° C. to a highstringency of about 0.2×SSC at 50° C. In addition, the temperature inthe wash step can be increased from low stringency conditions at roomtemperature, about 22° C., to high stringency conditions at about 65° C.

[0062] Nucleic acids, having a sequence that differs from the nucleotidesequence shown in SEQ ID No: 1 due to degeneracy in the genetic code arealso within the scope of the invention. Such nucleic acids encodefunctionally equivalent peptides (i.e., a peptide having a biologicalactivity of a ptc-2 polypeptide) but differ in sequence from thesequence shown in the sequence listing due to degeneracy in the geneticcode. For example, a number of amino acids are designated by more thanone triplet. Codons that specify the same amino acid, or synonyms (forexample, CAU and CAC each encode histidine) may result in “silent”mutations which do not affect the amino acid sequence of a ptc-2polypeptide. However, it is expected that DNA sequence polymorphismsthat do lead to changes in the amino acid sequences of the subject ptc-2polypeptides will exist among humans. One skilled in the art willappreciate that these variations in one or more nucleotides (up to about3-5% of the nucleotides) of the nucleic acids encoding polypeptideshaving an activity of a ptc-2 polypeptide may exist among individuals ofa given species due to natural allelic variation.

[0063] As used herein, a ptc-2 gene fragment refers to a nucleic acidhaving fewer nucleotides than the nucleotide sequence encoding theentire mature form of a ptc-2 protein yet which (preferably) encodes apolypeptide which retains some biological activity of the full lengthprotein. Fragment sizes contemplated by the present invention include,for example, 5, 10, 25, 50, 75, 100, or 200 amino acids in length. In apreferred embodiment of a truncated receptor, the polypeptide willinclude all or a sufficient portion of the ligand domain to bind to ahedgehog polypeptide.

[0064] As indicated by the examples set out below, ptc-2protein-encoding nucleic acids can be obtained from mRNA present incells of metazoan organisms. It should also be possible to obtainnucleic acids encoding ptc-2 polypeptides of the present invention fromgenomic DNA from both adults and embryos. For example, a gene encoding aptc-2 protein can be cloned from either a cDNA or a genomic library inaccordance with protocols described herein, as well as those generallyknown to persons skilled in the art. A cDNA encoding a ptc-2 protein canbe obtained by isolating total mRNA from a cell, such as a mammaliancell, e.g. a human cell, as desired. Double stranded cDNAs can beprepared from the total mRNA, and subsequently inserted into a suitableplasmid or bacteriophage vector using any one of a number of knowntechniques. The gene encoding a ptc-2 protein can also be cloned usingestablished polymerase chain reaction techniques in accordance with thenucleotide sequence information provided by the invention. The nucleicacid of the invention can be DNA or RNA. A preferred nucleic acid is acDNA including a nucleotide sequence represented by SEQ ID No: 1.

[0065] Another aspect of the invention relates to the use of theisolated nucleic acid in “antisense” therapy. As used herein,“antisense” therapy refers to administration or in situ generation ofoligonucleotide probes or their derivatives which specifically hybridize(e.g. binds) under cellular conditions, with the cellular mRNA and/orgenomic DNA encoding a subject ptc-2 protein so as to inhibit expressionof that protein, e.g. by inhibiting transcription and/or translation.The binding may be by conventional base pair complementarity, or, forexample, in the case of binding to DNA duplexes, through specificinteractions in the major groove of the double helix. In general,“antisense” therapy refers to the range of techniques generally employedin the art, and includes any therapy which relies on specific binding tooligonucleotide sequences.

[0066] An antisense construct of the present invention can be delivered,for example, as an expression plasmid which, when transcribed in thecell, produces RNA which is complementary to at least a unique portionof the cellular mRNA which encodes a ptc-2 protein. Alternatively, theantisense construct is an oligonucleotide probe which is generated exvivo and which, when introduced into the cell causes inhibition ofexpression by hybridizing with the mRNA and/or genomic sequences of aptc-2 gene. Such oligonucleotide probes are preferably modifiedoligonucleotides which are resistant to endogenous nucleases, e.g.exonucleases and/or endonucleases, and are therefore stable in vivo.Exemplary nucleic acid molecules for use as antisense oligonucleotidesare phosphoramidite, phosphorothioate and methylphosphonate analogs ofDNA (see also U.S. Pat. Nos. 5,176,996; 5,264,564; and 5,256,775), orpeptide nucleic acids (PNAs). Additionally, general approaches toconstructing oligomers useful in antisense therapy have been reviewed,for example, by Van der Krol et al. (1988) Biotechniques 6:958-976; andStein et al. (1988) Cancer Res 48:2659-2668.

[0067] Accordingly, the modified oligomers of the invention are usefulin therapeutic, diagnostic, and research contexts. In therapeuticapplications, the oligomers are utilized in a manner appropriate forantisense therapy in general. For such therapy, the oligomers of theinvention can be formulated for a variety of routes of administration,including systemic and topical or localized administration. Techniquesand formulations generally may be found in Remington's PharmaceuticalSciences, Meade Publishing Co., Easton, Pa. For systemic administration,injection is preferred, including intramuscular, intravenous,intraperitoneal, and subcutaneous. For injection, the oligomers of theinvention can be formulated in liquid solutions, preferably inphysiologically compatible buffers such as Hank's solution or Ringer'ssolution. In addition, the oligomers may be formulated in solid form andredissolved or suspended immediately prior to use. Lyophilized forms arealso included.

[0068] Systemic administration can also be by transmucosal ortransdermal means, or the compounds can be administered orally. Fortransmucosal or transdermal administration, penetrants appropriate tothe barrier to be permeated are used in the formulation. Such penetrantsare generally known in the art, and include, for example, fortransmucosal administration bile salts and fusidic acid derivatives. Inaddition, detergents may be used to facilitate permeation. Transmucosaladministration may be through nasal sprays or using suppositories. Fororal administration, the oligomers are formulated into conventional oraladministration forms such as capsules, tablets, and tonics. For topicaladministration, the oligomers of the invention are formulated intoointments, salves, gels, or creams as generally known in the art.

[0069] In addition to use in therapy, the oligomers of the invention maybe used as diagnostic reagents to detect the presence or absence of thetarget DNA or RNA sequences to which they specifically bind. Suchdiagnostic tests are described in further detail below.

[0070] Likewise, the antisense constructs of the present invention, byantagonizing the normal biological activity of a ptc-2 protein, e.g., byreducing the level of its expression, can be used in the manipulation oftissue, e.g. tissue maintenance, differentiation or growth, both in vivoand ex vivo.

[0071] Furthermore, the anti-sense techniques (e.g. microinjection ofantisense molecules, or transfection with plasmids whose transcripts areanti-sense with regard to a ptc-2 mRNA or gene sequence) can be used toinvestigate the role of ptc-2 in developmental events, as well as thenormal cellular function of ptc-2 in adult tissue. Such techniques canbe utilized in cell culture, but can also be used in the creation oftransgenic animals (described infra).

[0072] This invention also provides expression vectors containing anucleic acid encoding a ptc-2 polypeptide, operably linked to at leastone transcriptional regulatory sequence. Operably linked is intended tomean that the nucleotide sequence is linked to a regulatory sequence ina manner which allows expression of the nucleotide sequence. Regulatorysequences are art-recognized and are selected to direct expression ofthe subject ptc-2 proteins. Accordingly, the term transcriptionalregulatory sequence includes promoters, enhancers and other expressioncontrol elements. Such regulatory sequences are described in Goeddel;Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990). For instance, any of a wide variety ofexpression control sequences, sequences that control the expression of aDNA sequence when operatively linked to it, may be used in these vectorsto express DNA sequences encoding ptc-2 polypeptides of this invention.Such useful expression control sequences, include, for example, a viralLTR, such as the LTR of the Moloney murine leukemia virus, the early andlate promoters of SV40, adenovirus or cytomegalovirus immediate earlypromoter, the lac system, the trp system, the TAC or TRC system, T7promoter whose expression is directed by T7 RNA polymerase, the majoroperator and promoter regions of phage λ, the control regions for fdcoat protein, the promoter for 3-phosphoglycerate kinase or otherglycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, thepromoters of the yeast α-mating factors, the polyhedron promoter of thebaculovirus system and other sequences known to control the expressionof genes of prokaryotic or eukaryotic cells or their viruses, andvarious combinations thereof. It should be understood that the design ofthe expression vector may depend on such factors as the choice of thehost cell to be transformed and/or the type of protein desired to beexpressed.

[0073] Moreover, the vector's copy number, the ability to control thatcopy number and the expression of any other proteins encoded by thevector, such as antibiotic markers, should also be considered. In oneembodiment, the expression vector includes a recombinant gene encoding apolypeptide having an agonistic activity of a subject ptc-2 polypeptide,or alternatively, encoding a polypeptide which is an antagonistic formof the ptc-2 protein. An exemplary ptc-2 polypeptide of the presentinvention is a soluble truncated form of the protein which retains theligand binding domain, e.g., retains the ability to bind to hedgehogpolypeptides. Such expression vectors can be used to transfect cells andthereby produce polypeptides, including fusion proteins, encoded bynucleic acids as described herein.

[0074] Moreover, the gene constructs of the present invention can alsobe used as a part of a gene therapy protocol to deliver nucleic acids,e.g., encoding either an agonistic or antagonistic form of a subjectptc-2 proteins or an antisense molecule described above. Thus, anotheraspect of the invention features expression vectors for in vivo or invitro transfection and expression of a ptc-2 polypeptide or antisensemolecule in particular cell types so as to reconstitute the function of,or alternatively, abrogate all or a portion of the biological functionof ptc-2-induced transcription in a tissue in which thenaturally-occurring form of the protein is misexpressed (or has beendisrupted); or to deliver a form of the protein which alters maintenanceor differentiation of tissue, or which inhibits neoplastic orhyperplastic proliferation.

[0075] Expression constructs of the subject ptc-2 polypeptides, as wellas antisense constructs, may be administered in any biologicallyeffective carrier, e.g. any formulation or composition capable ofeffectively delivering the recombinant gene to cells in vivo. Approachesinclude insertion of the subject gene in viral vectors includingrecombinant retroviruses, adenovirus, adeno-associated virus, and herpessimplex virus-1, or recombinant bacterial or eukaryotic plasmids. Viralvectors transfect cells directly; plasmid DNA can be delivered with thehelp of, for example, cationic liposomes (lipofectin) or derivatized(e.g. antibody conjugated), polylysine conjugates, gramacidin S,artificial viral envelopes or other such intracellular carriers, as wellas direct injection of the gene construct or CaPO₄ precipitation carriedout in vivo. It will be appreciated that because transduction ofappropriate target cells represents the critical first step in genetherapy, choice of the particular gene delivery system will depend onsuch factors as the phenotype of the intended target and the route ofadministration, e.g. locally or systemically. Furthermore, it will berecognized that the particular gene construct provided for in vivotransduction of ptc-2 expression are also useful for in vitrotransduction of cells, such as for use in the ex vivo tissue culturesystems described below.

[0076] A preferred approach for in vivo introduction of nucleic acidinto a cell is by use of a viral vector containing nucleic acid, e.g. acDNA encoding the particular ptc-2 polypeptide desired. Infection ofcells with a viral vector has the advantage that a large proportion ofthe targeted cells can receive the nucleic acid. Additionally, moleculesencoded within the viral vector, e.g., by a cDNA contained in the viralvector, are expressed efficiently in cells which have taken up viralvector nucleic acid. Retrovirus vectors, adenovirus vectors andadeno-associated virus vectors are exemplary recombinant gene deliverysystem for the transfer of exogenous genes in vivo, particularly intohumans. These vectors provide efficient delivery of genes into cells,and the transferred nucleic acids are stably integrated into thechromosomal DNA of the host.

[0077] In addition to viral transfer methods, such as those illustratedabove, non-viral methods can also be employed to cause expression of asubject ptc-2 polypeptide in the tissue of an animal. Most nonviralmethods of gene transfer rely on normal mechanisms used by mammaliancells for the uptake and intracellular transport of macromolecules. Inpreferred embodiments, non-viral gene delivery systems of the presentinvention rely on endocytic pathways for the uptake of the subject ptc-2polypeptide gene by the targeted cell. Exemplary gene delivery systemsof this type include liposomal derived systems, poly-lysine conjugates,and artificial viral envelopes.

[0078] In clinical settings, the gene delivery systems for thetherapeutic ptc-2 gene can be introduced into a patient-animal by any ofa number of methods, each of which is familiar in the art. For instance,a pharmaceutical preparation of the gene delivery system can beintroduced systemically, e.g. by intravenous injection, and specifictransduction of the protein in the target cells occurs predominantlyfrom specificity of transfection provided by the gene delivery vehicle,cell-type or tissue-type expression due to the transcriptionalregulatory sequences controlling expression of the receptor gene, or acombination thereof. In other embodiments, initial delivery of therecombinant gene is more limited with introduction into the animal beingquite localized. For example, the gene delivery vehicle can beintroduced by catheter (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (e.g. Chen et al. (1994) PNAS 91: 3054-3057). A ptc-2 gene canbe delivered in a gene therapy construct by electroporation usingtechniques described, for example, by Dev et al. ((1994) Cancer TreatRev 20:105-115).

[0079] The pharmaceutical preparation of the gene therapy construct canconsist essentially of the gene delivery system in an acceptablediluent, or can comprise a slow release matrix in which the genedelivery vehicle is imbedded. Alternatively, where the complete genedelivery system can be produced intact from recombinant cells, e.g.retroviral vectors, the pharmaceutical preparation can comprise one ormore cells which produce the gene delivery system.

[0080] In yet another embodiment, the subject invention provides a “geneactivation” construct which, by homologous recombination with a genomicDNA, alters the transcriptional regulatory sequences of an endogenousptc-2 gene. For instance, the gene activation construct can replace theendogenous promoter of a ptc-2 gene with a heterologous promoter, e.g.,one which causes constitutive expression of the ptc-2 gene or whichcauses inducible expression of the gene under conditions different fromthe normal expression pattern of ptc-2. A variety of different formatsfor the gene activation constructs are available. See, for example, theTranskaryotic Therapies, Inc. PCT publications W093/09222, W095/31560,W096/2941 1, W095/31560 and W094/12650.

[0081] In preferred embodiments, the nucleotide sequence used as thegene activation construct can be comprised of (1) DNA from some portionof the endogenous ptc-2 gene (exon sequence, intron sequence, promotersequences, etc.) which direct recombination and (2) heterologoustranscriptional regulatory sequence(s) which is to be operably linked tothe coding sequence for the genomic ptc-2 gene upon recombination of thegene activation construct. For use in generating cultures of ptc-2producing cells, the construct may further include a reporter gene todetect the presence of the knockout construct in the cell.

[0082] The gene activation construct is inserted into a cell, andintegrates with the genomic DNA of the cell in such a position so as toprovide the heterologous regulatory sequences in operative associationwith the native ptc-2 gene. Such insertion occurs by homologousrecombination, i.e., recombination regions of the activation constructthat are homologous to the endogenous ptc-2 gene sequence hybridize tothe genomic DNA and recombine with the genomic sequences so that theconstruct is incorporated into the corresponding position of the genomicDNA.

[0083] The terms “recombination region” or “targeting sequence” refer toa segment (i.e., a portion) of a gene activation construct having asequence that is substantially identical to or substantiallycomplementary to a genomic gene sequence, e.g., including 5′ flankingsequences of the genomic gene, and can facilitate homologousrecombination between the genomic sequence and the targeting transgeneconstruct.

[0084] As used herein, the term “replacement region” refers to a portionof a activation construct which becomes integrated into an endogenouschromosomal location following homologous recombination between arecombination region and a genomic sequence.

[0085] The heterologous regulatory sequences, e.g., which are providedin the replacement region, can include one or more of a varietyelements, including: promoters (such as constitutive or induciblepromoters), enhancers, negative regulatory elements, locus controlregions, transcription factor binding sites, or combinations thereof.Promoters/enhancers which may be used to control the expression of thetargeted gene in vivo include, but are not limited to, thecytomegalovirus (CMV) promoter/enhancer (Karasuyama et al., 1989, J.Exp. Med., 169:13), the human β-actin promoter (Gunning et al. (1987)PNAS 84:4831-4835), the glucocorticoid-inducible promoter present in themouse mammary tumor virus long terminal repeat (MMTV LTR) (Klessig etal. (1984) Mol. Cell Biol. 4:1354-1362), the long terminal repeatsequences of Moloney murine leukemia virus (MULV LTR) (Weiss et al.(1985) RNA Tumor Viruses, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.), the SV40 early or late region promoter (Bernoist et al.(1981) Nature 290:304-310; Templeton et al. (1984) Mol. Cell Biol.,4:817; and Sprague et al. (1983) J. Virol., 45:773), the promotercontained in the 3′ long terminal repeat of Rous sarcoma virus (RSV)(Yamamoto et al., 1980, Cell, 22:787-797), the herpes simplex virus(HSV) thymidine kinase promoter/enhancer (Wagner et al. (1981) PNAS82:3567-71), and the herpes simplex virus LAT promoter (Wolfe et al.(1992) Nature Genetics, 1:379-384).

[0086] In still other embodiments, the replacement region merely deletesa negative transcriptional control element of the native gene, e.g., toactivate expression, or ablates a positive control element, e.g., toinhibit expression of the targeted gene.

[0087] Another aspect of the present invention concerns recombinantforms of the ptc-2 proteins. Recombinant polypeptides preferred by thepresent invention, in addition to native ptc-2 proteins, are at least85% or 90% homologous, more preferably at least 95% homologous and mostpreferably at least 98% homologous with an amino acid sequencerepresented by SEQ ID No: 2. Such polypeptides, as described above,include various truncated forms of the protein.

[0088] The term “recombinant ptc-2 polypeptide” refers to a polypeptidewhich is produced by recombinant DNA techniques, wherein generally, DNAencoding a ptc-2 polypeptide is inserted into a suitable expressionvector which is in turn used to transform a host cell to produce theheterologous protein. Moreover, the phrase “derived from”, with respectto a recombinant ptc-2 gene, is meant to include within the meaning of“recombinant protein” those proteins having an amino acid sequence of anative ptc-2 protein, or an amino acid sequence similar thereto which isgenerated by mutations including substitutions and deletions (includingtruncation) of a naturally occurring form of the protein.

[0089] The present invention further pertains to recombinant forms ofthe subject ptc-2 polypeptides which are encoded by genes derived from amammal (e.g. a human), reptile or amphibian and which have amino acidsequences evolutionarily related to the ptc-2 protein represented in SEQID No: 2. Such recombinant ptc-2 polypeptides preferably are capable offunctioning in one of either role of an agonist or antagonist of atleast one biological activity of a wild-type (“authentic”) ptc-2 proteinof the appended sequence listing. The term “evolutionarily related to”,with respect to amino acid sequences of ptc-2 proteins, refers to bothpolypeptides having amino acid sequences which have arisen naturally,and also to mutational variants of ptc-2 polypeptides which are derived,for example, by combinatorial mutagenesis.

[0090] The present invention also provides methods of producing thesubject ptc-2 polypeptides. For example, a host cell transfected with anucleic acid vector directing expression of a nucleotide sequenceencoding the subject polypeptides can be cultured under appropriateconditions to allow expression of the peptide to occur. A cell cultureincludes host cells, media and other byproducts. Suitable media for cellculture are well known in the art. The recombinant ptc-2 polypeptide(e.g., soluble fragments) can be isolated from cell culture medium, hostcells, or both using techniques known in the art for purifying proteinsincluding ion-exchange chromatography, gel filtration chromatography,ultrafiltration, electrophoresis, and immunoaffinity purification withantibodies specific for such peptide. In other embodiments, therecombinant ptc-2 polypeptide is obtained from membrane preparations ofthe host cells.

[0091] This invention also pertains to a host cell transfected toexpress recombinant forms of the subject ptc-2 polypeptides. The hostcell may be any eukaryotic or prokaryotic cell. Thus, a nucleotidesequence derived from the cloning of ptc-2 proteins, encoding all or aselected portion of a full-length protein, can be used to produce arecombinant form of a ptc-2 polypeptide via microbial or eukaryoticcellular processes. Ligating the polynucleotide sequence into a geneconstruct, such as an expression vector, and transforming ortransfecting into hosts, either eukaryotic (yeast, avian, insect ormammalian) or prokaryotic (bacterial cells), are standard proceduresused in producing other well-known proteins, e.g. hedgehog proteins,TGFβ proteins, as well as a wide range of receptors. Similar procedures,or modifications thereof, can be employed to prepare recombinant ptc-2polypeptides by microbial means or tissue-culture technology in accordwith the subject invention.

[0092] The recombinant ptc-2 genes can be produced by ligating nucleicacid encoding a ptc-2 polypeptide into a vector suitable for expressionin either prokaryotic cells, eukaryotic cells, or both. Expressionvectors for production of recombinant forms of the subject ptc-2polypeptides include plasmids and other vectors. For instance, suitablevectors for the expression of a ptc-2 polypeptide include plasmids ofthe types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derivedplasmids, pBTac-derived plasmids and pUC-derived plasmids for expressionin prokaryotic cells, such as E. coli.

[0093] A number of vectors exist for the expression of recombinantproteins in yeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, andYRP17 are cloning and expression vehicles useful in the introduction ofgenetic constructs into S. cerevisiae (see, for example, Broach et al.(1983) in Experimental Manipulation of Gene Expression, ed. M. InouyeAcademic Press, p. 83, incorporated by reference herein). These vectorscan replicate in E. coli due the presence of the pBR322 ori, and in S.cerevisiae due to the replication determinant of the yeast 2 micronplasmid. In addition, drug resistance markers such as ampicillin can beused. In an illustrative embodiment, a ptc-2 polypeptide is producedrecombinantly utilizing an expression vector generated by sub-cloningthe coding sequence of a ptc-2 gene represented in SEQ ID No: 1.

[0094] The preferred mammalian expression vectors contain bothprokaryotic sequences, to facilitate the propagation of the vector inbacteria, and one or more eukaryotic transcription units that areexpressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV,pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo andpHyg derived vectors are examples of mammalian expression vectorssuitable for transfection of eukaryotic cells. Some of these vectors aremodified with sequences from bacterial plasmids, such as pBR322, tofacilitate replication and drug resistance selection in both prokaryoticand eukaryotic cells. Alternatively, derivatives of viruses such as thebovine papillomavirus (BPV-1), or Epstein-Barr virus (pHEBo,pREP-derived and p205) can be used for transient expression of proteinsin eukaryotic cells. The various methods employed in the preparation ofthe plasmids and transformation of host organisms are well known in theart. For other suitable expression systems for both prokaryotic andeukaryotic cells, as well as general recombinant procedures, seeMolecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory Press: 1989) Chapters 16 and17.

[0095] In some instances, it may be desirable to express the recombinantplc-2 polypeptide by the use of a baculovirus expression system.Examples of such baculovirus expression systems include pVL-derivedvectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors(such as pAcUWI), and pBlueBac-derived vectors (such as the β-galcontaining pBlueBac III).

[0096] When it is desirable to express only a portion of a ptc-2protein, such as a form lacking a portion of the N-terminus, i.e. atruncation mutant which lacks the signal peptide, it may be necessary toadd a start codon (ATG) to the oligonucleotide fragment containing thedesired sequence to be expressed. It is well known in the art that amethionine at the N-terminal position can be enzymatically cleaved bythe use of the enzyme methionine aminopeptidase (MAP). MAP has beencloned from E. coli (Ben-Bassat et al. (1987) J. Bacteriol. 169:751-757)and Salmonella typhimurium and its in vitro activity has beendemonstrated on recombinant proteins (Miller et al. (1987) PNAS84:2718-1722). Therefore, removal of an N-terminal methionine, ifdesired, can be achieved either in vivo by expressing ptc-2-derivedpolypeptides in a host which produces MAP (e.g., E. coli or CM89 or S.cerevisiae), or in vitro by use of purified MAP (e.g., procedure ofMiller et al., supra).

[0097] Alternatively, the coding sequences for the polypeptide can beincorporated as a part of a fusion gene including a nucleotide sequenceencoding a different polypeptide. This type of expression system can beuseful under conditions where it is desirable to produce an immunogenicfragment of a ptc-2 protein. For example, the VP6 capsid protein ofrotavirus can be used as an immunologic carrier protein for portions ofthe ptc-2 polypeptide, either in the monomeric form or in the form of aviral particle. The nucleic acid sequences corresponding to the portionof a subject ptc-2 protein to which antibodies are to be raised can beincorporated into a fusion gene construct which includes codingsequences for a late vaccinia virus structural protein to produce a setof recombinant viruses expressing fusion proteins comprising ptc-2epitopes as part of the virion. It has been demonstrated with the use ofimmunogenic fusion proteins utilizing the Hepatitis B surface antigenfusion proteins that recombinant Hepatitis B virions can be utilized inthis role as well. Similarly, chimeric constructs coding for fusionproteins containing a portion of a ptc-2 protein and the polioviruscapsid protein can be created to enhance immunogenicity of the set ofpolypeptide antigens (see, for example, EP Publication No: 0259149; andEvans et al. (1989) Nature 339:385; Huang et al. (1988) J. Virol.62:3855; and Schlienger et al. (1992) J. Virol. 66:2).

[0098] The Multiple Antigen Peptide system for peptide-basedimmunization can also be utilized to generate an immunogen, wherein adesired portion of a ptc-2 polypeptide is obtained directly fromorgano-chemical synthesis of the peptide onto an oligomeric branchinglysine core (see, for example, Posnett et al. (1988) JBC 263:1719 andNardelli et al. (1992) J. Immunol. 148:914). Antigenic determinants ofptc-2 proteins can also be expressed and presented by bacterial cells.

[0099] In addition to utilizing fusion proteins to enhanceimmunogenicity, it is widely appreciated that fusion proteins can alsofacilitate the expression of proteins, and accordingly, can be used inthe expression of the ptc-2 polypeptides of the present invention,particularly truncated forms of the ptc-2 protein. For example, ptc-2polypeptides can be generated as glutathione-S-transferase (GST-fusion)proteins. Such GST-fusion proteins can enable easy purification of theptc-2 polypeptide, as for example by the use of glutathione-derivatizedmatrices (see, for example, Current Protocols in Molecular Biology, eds.Ausubel et al. (N.Y.: John Wiley & Sons, 1991)).

[0100] In another embodiment, a fusion gene coding for a purificationleader sequence, such as a poly-(His)/enterokinase cleavage sitesequence at the N-terminus of the desired portion of the recombinantprotein, can allow purification of the expressed fusion protein byaffinity chromatography using a Ni2+ metal resin. The purificationleader sequence can then be subsequently removed by treatment withenterokinase to provide the purified protein (e.g., see Hochuli et al.(1987) J. Chromatography 411:177; and Janknecht et al. PNAS 88:8972).

[0101] Techniques for making fusion genes are known to those skilled inthe art. Essentially, the joining of various DNA fragments coding fordifferent polypeptide sequences is performed in accordance withconventional techniques, employing blunt-ended or stagger-ended terminifor ligation, restriction enzyme digestion to provide for appropriatetermini, filling-in of cohesive ends as appropriate, alkalinephosphatase treatment to avoid undesirable joining, and enzymaticligation. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed togenerate a chimeric gene sequence (see, for example, Current Protocolsin Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).

[0102] The ptc-2 polypeptides may also be chemically modified to createptc-2 derivatives by forming covalent or aggregate conjugates with otherchemical moieties, such as glycosyl groups, lipids, cholesterol,phosphate, acetyl groups and the like. Covalent derivatives of ptc-2proteins can be prepared by linking the chemical moieties to functionalgroups on amino acid sidechains of the protein or at the N-terminus orat the C-terminus of the polypeptide.

[0103] The present invention also makes available isolated ptc-2polypeptides which are isolated from, or otherwise substantially free ofother cellular proteins, especially receptors and/or other inductivepolypeptides which may normally be associated with the ptc-2 polypeptide(such as smoothened). The term “substantially free of other cellularproteins” (also referred to herein as “contaminating proteins”) or“substantially pure or purified preparations” are defined asencompassing preparations of ptc-2 polypeptides having less than 20% (bydry weight) contaminating protein, and preferably having less than 5%contaminating protein. Functional forms of the subject polypeptides canbe prepared, for the first time, as purified preparations by using acloned gene as described herein. By “purified”, it is meant, whenreferring to a peptide or DNA or RNA sequence, that the indicatedmolecule is present in the substantial absence of other biologicalmacromolecules, such as other proteins. The term “purified” as usedherein preferably means at least 80% by dry weight, more preferably inthe range of 95-99% by weight, and most preferably at least 99.8% byweight, of biological macromolecules of the same type present (butwater, buffers, and other small molecules, especially molecules having amolecular weight of less than 5000, can be present). The term “pure” asused herein preferably has the same numerical limits as “purified”immediately above. “Isolated” and “purified” do not encompass eithernatural materials in their native state or natural materials that havebeen separated into components (e.g., in an acrylamide gel) but notobtained either as pure (e.g. lacking contaminating proteins, orchromatography reagents such as denaturing agents and polymers, e.g.acrylamide or agarose) substances or solutions. In preferredembodiments, purified ptc-2 preparations will lack any contaminatingproteins from the same animal from that ptc-2 is normally produced, ascan be accomplished by recombinant expression of for example, amammalian ptc-2 protein in a yeast or bacterial cell.

[0104] As described above for recombinant polypeptides, isolated ptc-2polypeptides can include all or a portion of an amino acid sequencescorresponding to a ptc-2 polypeptide represented in SEQ ID No: 2 orhomologous sequences thereto.

[0105] Isolated peptidyl portions of ptc-2 proteins can also be obtainedby screening peptides recombinantly produced from the correspondingfragment of the nucleic acid encoding such peptides. In addition,fragments can be chemically synthesized using techniques known in theart such as conventional Merrifield solid phase f-Moc or t-Bocchemistry. For example, a ptc-2 polypeptide of the present invention maybe arbitrarily divided into fragments of desired length with no overlapof the fragments, or preferably divided into overlapping fragments of adesired length. The fragments can be produced (recombinantly or bychemical synthesis) and tested to identify those peptidyl fragmentswhich can function as either agonists or antagonists of a wild-type(e.g., “authentic”) ptc-2 protein. For example, Román et al. (1994) EurJ Biochem 222:65-73 describe the use of competitive-binding assays usingshort, overlapping synthetic peptides from larger proteins to identifybinding domains.

[0106] The recombinant ptc-2 polypeptides of the present invention alsoinclude homologs of the authentic ptc-2 proteins, such as versions ofthose protein which are resistant to proteolytic cleavage, as forexample, due to mutations which alter ubiquitination, prenylation or thelike, enzymatic release of the extracellular domain, or other enzymatictargeting associated with the protein.

[0107] Modification of the structure of the subject ptc-2 polypeptidescan be for such purposes as enhancing therapeutic or prophylacticefficacy, stability (e.g., ex vivo shelf life and resistance toproteolytic degradation in vivo), or post-translational modifications.Such modified peptides, when designed to retain at least one activity ofthe naturally-occurring form of the protein, or to produce specificantagonists thereof, are considered functional equivalents of the ptc-2polypeptides (though they may be agonistic or antagonistic of thebioactivities of the authentic protein). Such modified peptides can beproduced, for instance, by amino acid substitution, deletion, oraddition.

[0108] For example, it is reasonable to expect that an isolatedreplacement of a leucine with an isoleucine or valine, an aspartate witha glutamate, a threonine with a serine, or a similar replacement of anamino acid with a structurally related amino acid (i.e. isosteric and/orisoelectric mutations) will not have a major effect on the biologicalactivity of the resulting molecule. Conservative replacements are thosethat take place within a family of amino acids that are related in theirside chains. Genetically encoded amino acids are can be divided intofour families: (1) acidic=aspartate, glutamate; (2) basic=lysine,arginine, histidine; (3) nonpolar=alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan; and (4) unchargedpolar=glycine, asparagine, glutamine, cysteine, serine, threonine,tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimesclassified jointly as aromatic amino acids. In similar fashion, theamino acid repertoire can be grouped as (1) acidic=aspartate, glutamate;(2) basic=lysine, arginine histidine, (3) aliphatic=glycine, alanine,valine, leucine, isoleucine, serine, threonine, with serine andthreonine optionally be grouped separately as aliphatic-hydroxyl; (4)aromatic=phenylalanine, tyrosine, tryptophan; (5) amide=asparagine,glutamine; and (6) sulfur-containing=cysteine and methionine. (see, forexample, Biochemistry, 2nd ed., Ed. by L. Stryer, WH Freeman and Co.:1981). Whether a change in the amino acid sequence of a peptide resultsin a functional ptc-2 homolog (e.g. functional in the sense that theresulting polypeptide mimics or antagonizes the authentic form) can bereadily determined by assessing the ability of the variant peptide toproduce a response in cells in a fashion similar to the wild-typeprotein, or competitively inhibit such a response. Polypeptides in whichmore than one replacement has taken place can readily be tested in thesame manner.

[0109] This invention further contemplates a method for generating setsof combinatorial point mutants of the subject ptc-2 proteins as well astruncation mutants, and is especially useful for identifying potentialvariant sequences (e.g. homologs) that are functional in modulatingsignal transduction and/or ligand binding. The purpose of screening suchcombinatorial libraries is to generate, for example, novel ptc-2homologs which can act as either agonists or antagonist, oralternatively, possess novel activities all together. To illustrate,ptc-2 homologs can be engineered by the present method to provideselective, constitutive activation of hedgehog activity, oralternatively, to be dominant negative inhibitors of ptc-2-dependentsignal transduction. For instance, mutagenesis can provide ptc-2homologs which are able to bind extracellular ligands yet be unable tobind or signal through intracellular regulatory proteins.

[0110] In one aspect of this method, the amino acid sequences for apopulation of ptc-2 homologs from different species or other relatedproteins are aligned, preferably to promote the highest homologypossible. Such a population of variants can include, for example, ptc-2homologs from one or more species. Amino acids which appear at eachposition of the aligned sequences are selected to create a degenerateset of combinatorial sequences. In a preferred embodiment, thevariegated library of ptc-2 variants is generated by combinatorialmutagenesis at the nucleic acid level, and is encoded by a variegatedgene library. For instance, a mixture of synthetic oligonucleotides canbe enzymatically ligated into gene sequences such that the degenerateset of potential ptc-2 sequences are expressible as individualpolypeptides, or alternatively, as a set of larger fusion proteins (e.g.for phage display) containing the set of ptc-2 sequences therein.

[0111] There are many ways by which such libraries of potential ptc-2homologs can be generated from a degenerate oligonucleotide sequence.Chemical synthesis of a degenerate gene sequence can be carried out inan automatic DNA synthesizer, and the synthetic genes then ligated intoan appropriate expression vector. The purpose of a degenerate set ofgenes is to provide, in one mixture, all of the sequences encoding thedesired set of potential ptc-2 sequences. The synthesis of degenerateoligonucleotides is well known in the art (see for example, Narang, S A(1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc 3rdCleveland Sympos. Macromolecules, ed. A G Walton, Amsterdam: Elsevierpp273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura etal. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.Such techniques have been employed in the directed evolution of otherproteins (see, for example, Scott et al. (1990) Science 249:386-390;Roberts et al. (1992) PNAS 89:2429-2433; Devlin et al. (1990) Science249: 404-406; Cwirla et al. (1990) PNAS 87: 6378-6382; as well as U.S.Pat. Nos. 5,223,409, 5,198,346, and 5,096,815).

[0112] Likewise, a library of coding sequence fragments can be providedfor a ptc-2 clone in order to generate a variegated population of ptc-2fragments for screening and subsequent selection of bioactive fragments.A variety of techniques are known in the art for generating suchlibraries, including chemical synthesis. In one embodiment, a library ofcoding sequence fragments can be generated by (i) treating a doublestranded PCR fragment of a ptc-2 coding sequence with a nuclease underconditions wherein nicking occurs only about once per molecule; (ii)denaturing the double stranded DNA; (iii) renaturing the DNA to formdouble stranded DNA which can include sense/antisense pairs fromdifferent nicked products; (iv) removing single stranded portions fromreformed duplexes by treatment with S1 nuclease; and (v) ligating theresulting fragment library into an expression vector. By this exemplarymethod, an expression library can be derived which codes for N-terminal,C-terminal and internal fragments of various sizes.

[0113] A wide range of techniques are known in the art for screeninggene products of combinatorial libraries made by point mutations ortruncation, and for screening cDNA libraries for gene products having acertain property. Such techniques will be generally adaptable for rapidscreening of the gene libraries generated by the combinatorialmutagenesis of ptc-2 homologs. The most widely used techniques forscreening large gene libraries typically comprises cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates relatively easy isolation of the vector encodingthe gene whose product was detected.

[0114] In an exemplary embodiment, a library of extracellular fragmentsof ptc-2 variants are expressed as a fusion protein on the surface of aviral particle, and the viral particles panned on a hedgehog matrix. Forinstance, in the filamentous phage system, foreign peptide sequences canbe expressed on the surface of infectious phage, thereby conferring twosignificant benefits. First, since these phage can be applied toaffinity matrices at very high concentrations, a large number of phagecan be screened at one time. Second, since each infectious phagedisplays the combinatorial gene product on its surface, if a particularphage is recovered from an affinity matrix in low yield, the phage canbe amplified by another round of infection. The group of almostidentical E. coli filamentous phages M13, fd., and fl are most oftenused in phage display libraries, as either of the phage gIII or gVIIIcoat proteins can be used to generate fusion proteins without disruptingthe ultimate packaging of the viral particle (Ladner et al. PCTpublication WO 90/02909; Garrard et al., PCT publication WO 92/09690;Marks et al. (1992) J. Biol. Chem. 267:16007-16010; Griffiths et al.(1993) EMBO J 12:725-734; Clackson et al. (1991) Nature 352:624-628; andBarbas et al. (1992) PNAS 89:4457-4461). For example, the recombinantphage antibody system (RPAS, Pharmacia Catalog number 27-9400-01) can beeasily modified for use in expressing and screening ptc-2 combinatoriallibraries by panning on a matrix-immobilized hedgehog polypeptides toenrich for ptc-2 homologs with enhanced ability to bind the ligand.

[0115] In another embodiment, libraries of membrane-bound ptc-2 variantsare expressed in a population of cells, which are subsequently used in ahedgehog binding assay.

[0116] The invention also provides for reduction of the ptc-2 protein togenerate mimetics, e.g. peptide or non-peptide agents, which are able todisrupt a biological activity of a ptc-2 polypeptide of the presentinvention, e.g. as inhibitors of protein-protein interactions, such aswith ligand proteins. Thus, such mutagenic techniques as described aboveare also useful to map the determinants of the ptc-2 proteins whichparticipate in protein-protein interactions involved in, for example,interaction of the subject ptc-2 polypeptide with hedgehog polypeptides.Alternatively, a similar system can be used to derive fragments of ahedgehog protein which bind to a ptc-2 protein and competitively inhibitbinding of the full length hedgehog protein.

[0117] To further illustrate, the critical residues of either a ptc-2protein or a hedgehog protein which are involved in molecularrecognition of the other can be determined and used to generateptc-2-derived or hedgehog-derived peptidomimetics which competitivelyinhibit Hedgehog/ptc-2 protein interactions. By employing, for example,scanning mutagenesis to map the amino acid residues of a protein whichis involved in binding other proteins, peptidomimetic compounds can begenerated which mimic those residues which facilitate the interaction.Such mimetics may then be used to interfere with the normal function ofa ptc-2 protein (or its ligand). For instance, non-hydrolyzable peptideanalogs of such residues can be generated using benzodiazepine (e.g.,see Freidinger et al. in Peptides: Chemistry and Biology, G. R. Marshalled., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., seeHuffman et al. in Peptides: Chemistry and Biology, G. R. Marshall ed.,ESCOM Publisher: Leiden, Netherlands, 1988), substituted gamma lactamrings (Garvey et al. in Peptides: Chemistry and Biology, G. R. Marshalled., ESCOM Publisher: Leiden, Netherlands, 1988), keto-methylenepseudopeptides (Ewenson et al. (1986) J Med Chem 29:295; and Ewenson etal. in Peptides: Structure and Function (Proceedings of the 9th AmericanPeptide Symposium) Pierce Chemical Co. Rockland, Ill., 1985), b-turndipeptide cores (Nagai et al. (1985) Tetrahedron Lett 26:647; and Satoet al. (1986) J Chem Soc Perkin Trans 1:1231), and b-aminoalcohols(Gordon et al. (1985) Biochem Biophys Res Commun 126:419; and Dann etal. (1986) Biochem Biophys Res Commun 134:71).

[0118] Another aspect of the invention pertains to an antibodyspecifically reactive with a ptc-2 protein. For example, by usingimmunogens derived from a ptc-2 protein, e.g. based on the cDNAsequences, anti-protein/anti-peptide antisera or monoclonal antibodiescan be made by standard protocols (See, for example, Antibodies: ALaboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press:1988)). A mammal, such as a mouse, a hamster or rabbit can be immunizedwith an immunogenic form of the peptide (e.g., a ptc-2 polypeptide or anantigenic fragment which is capable of eliciting an antibody response).Techniques for conferring immunogenicity on a protein or peptide includeconjugation to carriers or other techniques well known in the art. Animmunogenic portion of a ptc-2 protein can be administered in thepresence of adjuvant. The progress of immunization can be monitored bydetection of antibody titers in plasma or serum. Standard ELISA or otherimmunoassays can be used with the immunogen as antigen to assess thelevels of antibodies. In a preferred embodiment, the subject antibodiesare immunospecific for antigenic determinants of a ptc-2 protein of aorganism, such as a mammal, e.g. antigenic determinants of a proteinrepresented by SEQ ID No: 2 or closely related homologs. In yet afurther preferred embodiment of the present invention, in order toprovide, for example, antibodies which are immuno-selective for discreteptc-2 homologs the anti-ptc-2 polypeptide antibodies do notsubstantially cross react (i.e. does not react specifically) with aprotein which is, for example, less than 85%, 90% or 95% homologous withthe selected ptc-2. By “not substantially cross react”, it is meant thatthe antibody has a binding affinity for a non-homologous protein whichis at least one order of magnitude, more preferably at least 2 orders ofmagnitude, and even more preferably at least 3 orders of magnitude lessthan the binding affinity of the antibody for the intended target ptc-2.

[0119] Following immunization of an animal with an antigenic preparationof a ptc-2 polypeptide, anti-ptc-2 antisera can be obtained and, ifdesired, polyclonal anti-ptc-2 antibodies isolated from the serum. Toproduce monoclonal antibodies, antibody-producing cells (lymphocytes)can be harvested from an immunized animal and fused by standard somaticcell fusion procedures with immortalizing cells such as myeloma cells toyield hybridoma cells. Such techniques are well known in the art, aninclude, for example, the hybridoma technique (originally developed byKohler and Milstein, (1975) Nature, 256: 495-497), the human B cellhybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc. pp. 77-96). Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with a ptc-2 polypeptideof the present invention and monoclonal antibodies isolated from aculture comprising such hybridoma cells.

[0120] The term antibody as used herein is intended to include fragmentsthereof which are also specifically reactive with a ptc-2 polypeptide.Antibodies can be fragmented using conventional techniques and thefragments screened for utility in the same manner as described above forwhole antibodies. For example, F(ab)₂ fragments can be generated bytreating antibody with pepsin. The resulting F(ab)₂ fragment can betreated to reduce disulfide bridges to produce Fab fragments. Theantibody of the present invention is further intended to includebispecific and chimeric molecules having affinity for a ptc-2 proteinconferred by at least one CDR region of the antibody.

[0121] Both monoclonal and polyclonal antibodies (Ab) directed againstauthentic ptc-2 polypeptides, or ptc-2 variants, and antibody fragmentssuch as Fab, F(ab)₂, Fv and scFv can be used to block the action of aptc-2 protein and allow the study of the role of these proteins in, forexample, differentiation of tissue. Experiments of this nature can aidin deciphering the role of ptc-2 proteins that may be involved incontrol of proliferation versus differentiation, e.g., in patterning andtissue formation.

[0122] Antibodies which specifically bind ptc-2 epitopes can also beused in immunohistochemical staining of tissue samples in order toevaluate the abundance and pattern of expression of each of the subjectptc-2 polypeptides. Anti-ptc-2 antibodies can be used diagnostically inimmuno-precipitation and immuno-blotting to detect and evaluate ptc-2protein levels in tissue as part of a clinical testing procedure. Forinstance, such measurements can be useful in predictive valuations ofthe onset or progression of proliferative or differentiative disorders.Likewise, the ability to monitor ptc-2 protein levels in an individualcan allow determination of the efficacy of a given treatment regimen foran individual afflicted with such a disorder. The level of ptc-2polypeptides may be measured from cells in bodily fluid, such as insamples of cerebral spinal fluid or amniotic fluid, or can be measuredin tissue, such as produced by biopsy. Diagnostic assays usinganti-ptc-2 antibodies can include, for example, immunoassays designed toaid in early diagnosis of a disorder, particularly ones which aremanifest at birth. Diagnostic assays using anti-ptc-2 polypeptideantibodies can also include immunoassays designed to aid in earlydiagnosis and phenotyping neoplastic or hyperplastic disorders.

[0123] Another application of anti-ptc-2 antibodies of the presentinvention is in the immunological screening of cDNA librariesconstructed in expression vectors such as λgt11, λgt18-23, λZAP, andλORF8. Messenger libraries of this type, having coding sequencesinserted in the correct reading frame and orientation, can producefusion proteins. For instance, λgt11 will produce fusion proteins whoseamino termini consist of β-galactosidase amino acid sequences and whosecarboxy termini consist of a foreign polypeptide. Antigenic epitopes ofa ptc-2 protein, e.g. orthologs of the ptc-2 protein from other species,can then be detected with antibodies, as, for example, reactingnitrocellulose filters lifted from infected plates with anti-ptc-2antibodies. Positive phage detected by this assay can then be isolatedfrom the infected plate. Thus, the presence of ptc-2 homologs can bedetected and cloned from other animals, as can alternate isoforms(including splicing variants) from humans.

[0124] Moreover, the nucleotide sequences determined from the cloning ofptc-2 genes from organisms will further allow for the generation ofprobes and primers designed for use in identifying and/or cloning ptc-2homologs in other cell types, e.g. from other tissues, as well as ptc-2homologs from other organisms. For instance, the present invention alsoprovides a probe/primer comprising a substantially purifiedoligonucleotide, which oligonucleotide comprises a region of nucleotidesequence that hybridizes under stringent conditions to at least 15consecutive nucleotides of sense or anti-sense sequence selected fromthe group consisting of SEQ ID No: 1 or naturally occurring mutantsthereof. For instance, primers based on the nucleic acid represented inSEQ ID No: 1, can be used in PCR reactions to clone ptc-2 homologs.Likewise, probes based on the subject ptc-2 sequences can be used todetect transcripts or genomic sequences encoding the same or homologousproteins. In preferred embodiments, the probe further comprises a labelgroup attached thereto and able to be detected, e.g. the label group isselected from amongst radioisotopes, fluorescent compounds, enzymes, andenzyme co-factors.

[0125] Such probes can also be used as a part of a diagnostic test kitfor identifying cells or tissue which misexpress a ptc-2 protein, suchas by measuring a level of a ptc-2-encoding nucleic acid in a sample ofcells from a patient-animal; e.g. detecting ptc-2 mRNA levels ordetermining whether a genomic ptc-2 gene has been mutated or deleted.

[0126] To illustrate, nucleotide probes can be generated from thesubject ptc-2 genes which facilitate histological screening of intacttissue and tissue samples for the presence (or absence) ofptc-2-encoding transcripts. Similar to the diagnostic uses of anti-ptc-2antibodies, the use of probes directed to ptc-2 messages, or to genomicptc-2 sequences, can be used for both predictive and therapeuticevaluation of allelic mutations which might be manifest in, for example,degenerative disorders marked by loss of particular cell-types,apoptosis, neoplastic and/or hyperplastic disorders (e.g. unwanted cellgrowth) or abnormal differentiation of tissue. Used in conjunction withimmunoassays as described above, the oligonucleotide probes can helpfacilitate the determination of the molecular basis for a developmentaldisorder which may involve some abnormality associated with expression(or lack thereof) of a ptc-2 protein. For instance, variation inpolypeptide synthesis can be differentiated from a mutation in a codingsequence.

[0127] Accordingly, the present method provides a method for determiningif a subject is at risk for a disorder characterized by aberrantapoptosis, cell proliferation and/or differentiation. In preferredembodiments, method can be generally characterized as comprisingdetecting, in a sample of cells from the subject, the presence orabsence of a genetic lesion characterized by at least one of (i) analteration affecting the integrity of a gene encoding a ptc-2-protein,or (ii) the mis-expression of the ptc-2 gene. To illustrate, suchgenetic lesions can be detected by ascertaining the existence of atleast one of (i) a deletion of one or more nucleotides from a ptc-2gene, (ii) an addition of one or more nucleotides to a ptc-2 gene, (iii)a substitution of one or more nucleotides of a ptc-2 gene, (iv) a grosschromosomal rearrangement of a ptc-2 gene, (v) a gross alteration in thelevel of a messenger RNA transcript of a ptc-2 gene, (vii) aberrantmodification of a ptc-2 gene, such as of the methylation pattern of thegenomic DNA, (vii) the presence of a non-wild type splicing pattern of amessenger RNA transcript of a ptc-2 gene, (viii) a non-wild type levelof a ptc-2-protein, and (ix) inappropriate post-translationalmodification of a ptc-2-protein. As set out below, the present inventionprovides a large number of assay techniques for detecting lesions in aptc-2 gene, and importantly, provides the ability to discern betweendifferent molecular causes underlying ptc-2-dependent aberrant cellgrowth, proliferation and/or differentiation.

[0128] In an exemplary embodiment, there is provided a nucleic acidcomposition comprising a (purified) oligonucleotide probe including aregion of nucleotide sequence which is capable of hybridizing to a senseor antisense sequence of a ptc-2 gene, such as represented by any one ofSEQ ID No: 1, or naturally occurring mutants thereof, or 5′ or 3′flanking sequences or intronic sequences naturally associated with thesubject ptc-2 genes or naturally occurring mutants thereof. The nucleicacid of a cell is rendered accessible for hybridization, the probe isexposed to nucleic acid of the sample, and the hybridization of theprobe to the sample nucleic acid is detected. Such techniques can beused to detect lesions at either the genomic or mRNA level, includingdeletions, substitutions, etc., as well as to determine mRNA transcriptlevels.

[0129] In certain embodiments, detection of the lesion comprisesutilizing the probe/primer in a polymerase chain reaction (PCR) (see,e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACEPCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g.,Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al.(1944) PNAS 91:360-364), the later of which can be particularly usefulfor detecting point mutations in the ptc-2 gene. In a merelyillustrative embodiment, the method includes the steps of (i) collectinga sample of cells from a patient, (ii) isolating nucleic acid (e.g.,genomic, mRNA or both) from the cells of the sample, (iii) contactingthe nucleic acid sample with one or more primers which specificallyhybridize to a ptc-2 gene under conditions such that hybridization andamplification of the ptc-2 gene (if present) occurs, and (iv) detectingthe presence or absence of an amplification product, or detecting thesize of the amplification product and comparing the length to a controlsample.

[0130] In still another embodiment, the level of a ptc-2-protein can bedetected by immunoassay. For instance, the cells of a biopsy sample canbe lysed, and the level of a ptc-2-protein present in the cell can bequantitated by standard immunoassay techniques. In yet another exemplaryembodiment, aberrant methylation patterns of a ptc-2 gene can bedetected by digesting genomic DNA from a patient sample with one or morerestriction endonucleases that are sensitive to methylation and forwhich recognition sites exist in the ptc-2 gene (including in theflanking and intronic sequences). See, for example, Buiting et al.(1994) Human Mol Genet 3:893-895. Digested DNA is separated by gelelectrophoresis, and hybridized with probes derived from, for example,genomic or cDNA sequences. The methylation status of the ptc-2 gene canbe determined by comparison of the restriction pattern generated fromthe sample DNA with that for a standard of known methylation.

[0131] Furthermore, by making available purified and recombinant ptc-2polypeptides, the present invention facilitates the development ofassays which can be used to screen for drugs which are either agonistsor antagonists of the normal cellular function of the subject ptc-2proteins, or of their role in the pathogenesis of cellular maintenance,differentiation and/or proliferation and disorders related thereto. In ageneral sense, the assay evaluates the ability of a test compound tomodulate binding between a ptc-2 polypeptide and a molecule, e.g., aligand such as a hedgehog protein, that interacts with the ptc-2polypeptide, or the ability of the test compound to induce intracellularsignals in a ptc-2-dependent manner. Exemplary compounds which can bescreened against such ptc-2-mediated interactions include peptides,nucleic acids, carbohydrates, small organic molecules, and naturalproduct extract libraries, such as isolated from animals, plants, fungusand/or microbes.

[0132] In many drug screening programs which test libraries of compoundsand natural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity and/or bioavailability of the test compoundcan be generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity with a ligand.Accordingly, in an exemplary screening assay of the present invention, areaction mixture is generated to include at least a ligand-bindingportion of a ptc-2 polypeptide, compound(s) of interest, and a “targetmolecule”, e.g., a protein, which interacts with the ptc-2 polypeptide.Exemplary target molecules are the hedgehog proteins. Detection andquantification of interaction of the ptc-2 polypeptide with the targetmolecule provides a means for determining a compound's efficacy atinhibiting (or potentiating) interaction between the ptc-2 and thetarget molecule. The efficacy of the compound can be assessed bygenerating dose response curves from data obtained using variousconcentrations of the test compound. Moreover, a control assay can alsobe performed to provide a baseline for comparison. In the control assay,interaction of the ptc-2 polypeptide and target molecule is quantitatedin the absence of the test compound.

[0133] Interaction between the ptc-2 polypeptide and the target moleculemay be detected by a variety of techniques. Modulation of the formationof complexes can be quantitated using, for example, detectably labeledproteins such as radiolabelled, fluorescently labeled, or enzymaticallylabeled polypeptides, by immunoassay, by chromatographic detection, orby detecting the intrinsic activity of the acetylase.

[0134] Typically, it will be desirable to immobilize either ptc-2 or thetarget molecule to facilitate separation of complexes from uncomplexedforms of one or both of the proteins, as well as to accommodateautomation of the assay. Binding of ptc-2 to the target molecule, in thepresence and absence of a candidate agent, can be accomplished in anyvessel suitable for containing the reactants. Examples includemicrotitre plates, test tubes, and micro-centrifuge tubes. In oneembodiment, a fusion protein can be provided which adds a domain thatallows the protein to be bound to a matrix. For example,glutathione-S-transferase/ptc-2 (GST/ptc-2) fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the cell lysates, e.g. an ³⁵S-labeled, and the testcompound, and the mixture incubated under conditions conducive tocomplex formation, e.g. at physiological conditions for salt and pH,though slightly more stringent conditions may be desired. Followingincubation, the beads are washed to remove any unbound label, and thematrix immobilized and radiolabel determined directly (e.g. beads placedin scintillant), or in the supernatant after the complexes aresubsequently dissociated. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level oftarget molecule found in the bead fraction quantitated from the gelusing standard electrophoretic techniques.

[0135] Other techniques for immobilizing proteins and other molecules onmatrices are also available for use in the subject assay. For instance,either ptc-2 or target molecule can be immobilized utilizing conjugationof biotin and streptavidin. For instance, biotinylated ptc-2 moleculescan be prepared from biotin-NHS (N-hydroxy-succinimide) using techniqueswell known in the art (e.g., biotinylation kit, Pierce Chemicals,Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96well plates (Pierce Chemical). Alternatively, antibodies reactive withptc-2, but which do not interfere with the interaction between the ptc-2and target molecule, can be derivatized to the wells of the plate, andptc-2 trapped in the wells by antibody conjugation. As above,preparations of an target molecule and a test compound are incubated inthe ptc-2-presenting wells of the plate, and the amount of complextrapped in the well can be quantitated. Exemplary methods for detectingsuch complexes, in addition to those described above for theGST-immobilized complexes, include immunodetection of complexes usingantibodies reactive with the target molecule, or which are reactive withptc-2 protein and compete with the target molecule; as well asenzyme-linked assays which rely on detecting an enzymatic activityassociated with the target molecule, either intrinsic or extrinsicactivity. In the instance of the latter, the enzyme can be chemicallyconjugated or provided as a fusion protein with the target molecule. Toillustrate, the target molecule can be chemically cross-linked orgenetically fused (if it is a polypeptide) with horseradish peroxidase,and the amount of polypeptide trapped in the complex can be assessedwith a chromogenic substrate of the enzyme, e.g. 3,3′-diamino-benzadineterahydrochloride or 4-chloro-1-napthol. Likewise, a fusion proteincomprising the polypeptide and glutathione-S-transferase can beprovided, and complex formation quantitated by detecting the GSTactivity using 1-chloro-2,4-dinitrobenzene (Habig et al (1974) J BiolChem 249:7130).

[0136] For processes which rely on immunodetection for quantitatingproteins trapped in the complex, antibodies against the protein, such asanti-ptc-2 antibodies, can be used. Alternatively, the protein to bedetected in the complex can be “epitope tagged” in the form of a fusionprotein which includes, in addition to the ptc-2 sequence, a secondpolypeptide for which antibodies are readily available (e.g. fromcommercial sources). For instance, the GST fusion proteins describedabove can also be used for quantification of binding using antibodiesagainst the GST moiety. Other useful epitope tags include myc-epitopes(e.g., see Ellison et al. (1991) J Biol Chem 266:21150-21157) whichincludes a 10-residue sequence from c-myc, as well as the pFLAG system(International Biotechnologies, Inc.) or the pEZZ-protein A system(Pharamacia, N.J.).

[0137] An exemplary drug screening assay of the present inventionincludes the steps of (a) forming a reaction mixture including: (i) ahedgehog polypeptide, (ii) a ptc-2 polypeptide, and (iii) a testcompound; and (b) detecting interaction of the hedgehog and ptc-2polypeptides. A statistically significant change (potentiation orinhibition) in the interaction of the hedgehog and ptc-2 polypeptides inthe presence of the test compound, relative to the interaction in theabsence of the test compound, indicates a potential agonist (mimetic orpotentiator) or antagonist (inhibitor) of hedgehog bioactivity for thetest compound. The reaction mixture can be a cell-free proteinpreparation, e.g., a reconstituted protein mixture or a cell lysate, orit can be a recombinant cell including a heterologous nucleic acidrecombinantly expressing the ptc-2 polypeptide.

[0138] Where the desired portion of the hh receptor (or other hedgehogbinding molecule) cannot be provided in soluble form, the cell-freesystem can be, e.g., a cell membrane preparation, a reconstitutedprotein mixture, or a liposome reconstituting the ptc-2 protein. Forinstance, the ptc-2 protein can be purified from detergent extracts fromboth authentic and recombinant origins can be reconstituted inartificial lipid vesicles (e.g. phosphatidylcholine liposomes) or incell membrane-derived vesicles (see, for example, Bear et al. (1992)Cell 68:809-818; Newton et al. (1983) Biochemistry 22:6110-6117; andReber et al. (1987) J Biol Chem 262:11369-11374). The lamellar structureand size of the resulting liposomes can be characterized using electronmicroscopy. External orientation of the ptc-2 protein in thereconstituted membranes can be demonstrated, for example, byimmunoelectron microscopy. The interaction of a hedgehog protein withliposomes containing such ptc-2 complexes and liposomes without theprotein, in the presence of candidate agents, can be compared in orderto identify potential modulators of the hedgehog-ptc-2 polypeptideinteraction. The reconstituted ptc-2 membrane systems can also includeother proteins involved in hedgehog binding, e.g., such as smoothened.

[0139] In yet another embodiment, the drug screening assay is derived toinclude a whole cell expressing a ptc-2 polypeptide. The ability of atest agent to alter the activity of the ptc-2 protein can be detected byanalysis of the recombinant cell. For example, agonists and antagonistsof the ptc-2 biological activity can by detected by scoring foralterations in growth or differentiation (phenotype) of the cell.General techniques for detecting each are well known, and will vary withrespect to the source of the particular reagent cell utilized in anygiven assay. For the cell-based assays, the recombinant cell ispreferably a metazoan cell, e.g., a mammalian cell, e.g., an insectcell, e.g., a xenopus cell, e.g., an oocyte. In other embodiments, thehedgehog receptor can be reconstituted in a yeast cell.

[0140] In an exemplary embodiment, a cell which expresses the ptc-2receptor, e.g., whether endogenous or heterologous, can be contactedwith a ligand of the ptc-2 receptor, e.g., a hedgehog protein, which iscapable of inducing signal transduction from the receptor, and theresulting signaling detected either at various points in the pathway, oron the basis of a phenotypic change to the reagent cell. In oneembodiment, the reagent cell is contacted with antibody which causescross-linking of the receptor, and the signal cascade induced by thatcross-linking is subsequently detected. A test compound which modulatesthat pathway, e.g., potentiates or inhibits, can be detected bycomparison with control experiments which either lack the receptor orlack the test compound. For example, visual inspection of the morphologyof the reagent cell can be used to determine whether the biologicalactivity of the targeted ptc-2 protein has been affected by the addedagent.

[0141] In addition to morphological studies, change(s) in the level ofan intracellular second messenger responsive to signaling by the ptc-2polypeptide can be detected. For example, in various embodiments theassay may assess the ability of test agent to cause changes inphosphorylation patterns, adenylate cyclase activity (cCAMP production),GTP hydrolysis, calcium mobilization, and/or phospholipid hydrolysis(IP₃, DAG production) upon receptor stimulation. By detecting changes inintracellular signals, such as alterations in second messengers or geneexpression, in cells contacted with a hedgehog polypeptide, candidateagonists and antagonists to ptc-2-dependent hedgehog signaling can beidentified.

[0142] The transduction of certain intracellular signals can beinitiated by the specific interaction of an hh polypeptide with ptc-2protein, while other signals can be indirectly altered by thatinteraction. In Drosophila, and presumptively in vertebrate cells aswell, a number of gene products, including ptc-2, patched, thetranscription factor cubitus interruptus (ci), the serine/threoninekinase fused (fu) and the gene products of costal-2, smoothened andsuppressor of fused, have been implicated as putative components ofhedgehog-dependent signal transduction pathways. The recent cloning ofvertebrate homologs of the drosophila genes suggests that the hedgehogsignaling pathway is highly conserved from drosophila to vertebratespecies. The activity of each of these proteins can be detected directly(such as the kinase activity of fused, or can detected indirectly bymonitoring the level of second messengers produced downstream in thesignal pathway.

[0143] To further illustrate, recent studies have implicated proteinkinase A (PKA) as a possible component of hedgehog signaling indrosophila and vertebrate organisms (Hammerschmidt et al. (1996) Genes &Dev 10:647). High PKA activity has been shown to antagonize hedgehogsignaling in these systems. Although it is unclear whether PKA actsdirectly downstream or in parallel with hedgehog signaling, it ispossible that hedgehog signaling occurring through a ptc-2 proteineffects inhibition of PKA activity. Thus, detection of PKA activityprovides a potential readout for the instant assays.

[0144] Binding of hedgehog to ptc-2 proteins may stimulate the activityof phospholipases. Inositol lipids can be extracted and analyzed usingstandard lipid extraction techniques. Water soluble derivatives of allthree inositol lipids (IP₁, IP₂, IP₃) can also be quantitated usingradiolabelling techniques or HPLC.

[0145] The mobilization of intracellular calcium or the influx ofcalcium from outside the cell may be a response to hedgehog stimulationor lack there of. Calcium flux in the reagent cell can be measured usingstandard techniques. The choice of the appropriate calcium indicator,fluorescent, bioluminescent, metallochromic, or Ca⁺⁺-sensitivemicroelectrodes depends on the cell type and the magnitude and timeconstant of the event under study (Borle (1990) Environ Health Perspect84:45-56). As an exemplary method of Ca⁺⁺ detection, cells could beloaded with the Ca⁺⁺ sensitive fluorescent dye fura-2 or indo-1, usingstandard methods, and any change in Ca⁺⁺ measured using a fluorometer.

[0146] In certain embodiments of the assay, it may be desirable toscreen for changes in cellular phosphorylation. As an example, thedrosophila gene fused (fu) which encodes a serine/threonine kinase hasbeen identified as a potential downstream target in hedgehog signaling.(Preat et al., 1990 Nature 347, 87-89; Therond et al. 1993, Mech. Dev.44. 65-80). The ability of compounds to modulate serine/threonine kinaseactivation could be screened using colony immunoblotting (Lyons andNelson (1984) PNAS 81:7426-7430) using antibodies against phosphorylatedserine or threonine residues. Reagents for performing such assays arecommercially available, for example, phosphoserine and phosphothreoninespecific antibodies which measure increases in phosphorylation of thoseresidues can be purchased from commercial sources.

[0147] The interaction of a hedgehog protein with a ptc-2 protein mayset in motion a cascade involving the activation and inhibition ofdownstream effectors, the ultimate consequence of which is, in someinstances, a detectable change in the transcription or translation of agene. Potential transcriptional targets of ptc-2-dependent hedgehogsignaling include the ptc-2 gene itself, the patched gene (Hidalgo andIngham (1990) Development 110, 291-301 ;Marigo et al. (1996) Development122:1225-1233), and the vertebrate homologs of the drosophila cubitusinterruptus (ci) gene, the GLI genes (Hui et al. (1994) Dev Biol162:402-413). Patched gene expression has been shown to be induced incells of the limb bud and the neural plate that are responsive to Shh.(Marigo et al. (1996) PNAS, in press; Marigo et al., supra). The GLIgenes encode putative transcription factors having zinc finger DNAbinding domains (Orenic et al. (1990) Genes & Dev 4:1053-1067; Kinzleret al. (1990) Mol Cell Biol 10:634-642). Transcription of the GLI genehas been reported to be upregulated in response to hedgehog in limbbuds, while transcription of the GLI3 gene is downregulated in responseto hedgehog induction (Marigo et al. (1996) Development 122:1225-1233).By selecting transcriptional regulatory sequences from such targetgenes, e.g. from Hip or GLI genes, that are responsible for the up- ordown-regulation of these genes in response to hedgehog induction, andoperatively linking such promoters to a reporter gene, the presentinvention provides a transcription based assay which is sensitive to theability of a specific test compound to influence hedgehog signalingpathways.

[0148] In an exemplary embodiment, the step of detecting interaction ofthe hedgehog and ptc-2 polypeptides comprises detecting, in a cell-basedassay, change(s) in the level of expression of a gene controlled by atranscriptional regulatory sequence responsive to signaling by the ptc-2polypeptide. Reporter gene based assays of this invention measure theend stage of the above described cascade of events, e.g.,transcriptional modulation. Accordingly, in practicing one embodiment ofthe assay, a reporter gene construct is inserted into the reagent cellin order to generate a detection signal dependent on hedgehog signaling.Expression of the reporter gene, thus, provides a valuable screeningtool for the development of compounds that act as agonists orantagonists of ptc-2-dependent hedgehog induction.

[0149] In practicing one embodiment of the assay, a reporter geneconstruct is inserted into the reagent cell in order to generate adetection signal dependent on second messengers generated byptc-2-dependent induction with a hedgehog protein. Typically, thereporter gene construct will include a reporter gene in operativelinkage with one or more transcriptional regulatory elements responsiveto the hedgehog activity, with the level of expression of the reportergene providing the hedgehog-dependent detection signal. The amount oftranscription from the reporter gene may be measured using any methodknown to those of skill in the art to be suitable. For example, mRNAexpression from the reporter gene may be detected using RNAse protectionor RNA-based PCR, or the protein product of the reporter gene may beidentified by a characteristic stain or an intrinsic activity. Theamount of expression from the reporter gene is then compared to theamount of expression in either the same cell in the absence of the testcompound or it may be compared with the amount of transcription in asubstantially identical cell that lacks the target receptor protein. Anystatistically or otherwise significant difference in the amount oftranscription indicates that the test compound has in some manneraltered the inductive activity of the hedgehog protein.

[0150] As described in further detail below, in preferred embodimentsthe gene product of the reporter is detected by an intrinsic activityassociated with that product. For instance, the reporter gene may encodea gene product that, by enzymatic activity, gives rise to a detectionsignal based on color, fluorescence, or luminescence. In other preferredembodiments, the reporter or marker gene provides a selective growthadvantage, e.g., the reporter gene may enhance cell viability, relieve acell nutritional requirement, and/or provide resistance to a drug. Manyreporter genes are known to those of skill in the art and others may beidentified or synthesized by methods known to those of skill in the art.A reporter gene includes any gene that expresses a detectable geneproduct, which may be RNA or protein.

[0151] Preferred reporter genes are those that are readily detectable.The reporter gene may also be included in the construct in the form of afusion gene with a gene that includes desired transcriptional regulatorysequences or exhibits other desirable properties. Examples of reportergenes include, but are not limited to CAT (chloramphenicol acetyltransferase) (Alton and Vapnek (1979), Nature 282: 864-869) luciferase,and other enzyme detection systems, such as beta-galactosidase; fireflyluciferase (deWet et al. (1987), Mol. Cell. Biol. 7:725-737); bacterialluciferase (Engebrecht and Silverman (1984), PNAS 1: 4154-4158; Baldwinet al. (1984), Biochemistry 23: 3663-3667); alkaline phosphatase (Toh etal. (1989) Eur. J. Biochem. 182: 231-238, Hall et al. (1983) J. Mol.Appl. Gen. 2: 101), human placental secreted alkaline phosphatase(Cullen and Malim (1992) Methods in Enzymol. 216:362-368).

[0152] Accordingly, yet another embodiment of the subject drug screeningassays of the present invention provides a recombinant cell, e.g., forcarrying out certain of the drug screening methods above, comprising:(i) an expressible recombinant gene encoding a heterologous ptc-2polypeptide whose signal transduction activity is modulated by bindingto a hedgehog protein; and (ii) a reporter gene construct containing areporter gene in operative linkage with one or more transcriptionalregulatory elements responsive to the signal transduction activity ofthe ptc-2 polypeptide. Still another aspect of the present inventionprovides a kit for screening test compounds to identify agents whichmodulate the binding of hedgehog proteins with a hedgehog receptor,including the above-referenced cell and a preparation of purifiedhedgehog polypeptide.

[0153] After identifying certain test compounds as potential modulatorsof one or more bioactivities of a ptc-2 protein (such as hedgehogbinding), the practitioner of the subject assay will continue to testthe efficacy and specificity of the selected compounds both in vitro andin vivo. Whether for subsequent in vivo testing, or for administrationto an animal as an approved drug, agents identified in the subject assaycan be formulated in pharmaceutical preparations for in vivoadministration to an animal, preferably a human.

[0154] Another aspect of the present invention relates to a method ofinducing and/or maintaining a differentiated state, enhancing survival,and/or inhibiting (or alternatively potentiating) proliferation of acell, by contacting the cells with an agent which modulatesptc-2-dependent signal transduction pathways. The subject method couldbe used to generate and/or maintain an array of different tissue both invitro and in vivo. A “ptc-2 therapeutic,” whether inhibitory orpotentiating with respect to modulating the activity of a ptc-2 protein,can be, as appropriate, any of the preparations described above,including isolated ptc-2 polypeptides (including both agonist andantagonist forms), gene therapy constructs, antisense molecules,peptidomimetics, or agents identified in the drug assays providedherein. In certain embodiments, soluble forms of the ptc-2 proteinincluding the extracellular ligand-binding domain of the receptor can beprovided as a means for antagonizing the binding of a ptc-2 ligand to acell-surface ptc-2 receptor. For instance, such forms of the receptorcan be used to antagonize the bioactivity of a ligand of the receptor.

[0155] The ptc-2 therapeutic compounds of the present invention arelikely to play an important role in the modulation of cellularproliferation and maintenance of, for example, neuronal, testicular,osteogenic or chondrogenic tissues during disease states. It will alsobe apparent that, by transient use of modulators of ptc-2 activities, invivo reformation of tissue can be accomplished, e.g. in the developmentand maintenance of organs such as ectodermal patterning, as well ascertain mesodermal and endodermal differentiation processes. Bycontrolling the proliferative and differentiative potential fordifferent cells, the subject ptc-2 therapeutics can be used to reforminjured tissue, or to improve grafting and morphology of transplantedtissue. For instance, ptc-2 antagonists and agonists can be employed ina differential manner to regulate different stages of organ repair afterphysical, chemical or pathological insult. The present method is alsoapplicable to cell culture techniques.

[0156] To further illustrate this aspect of the invention, in vitroneuronal culture systems have proved to be fundamental and indispensabletools for the study of neural development, as well as the identificationof neurotrophic factors such as nerve growth factor (NGF), ciliarytrophic factors (CNTF), and brain derived neurotrophic factor (BDNF).Once a neuronal cell has become terminally-differentiated it typicallywill not change to another terminally differentiated cell-type. However,neuronal cells can nevertheless readily lose their differentiated state.This is commonly observed when they are grown in culture from adulttissue, and when they form a blastema during regeneration. The presentmethod provides a means for ensuring an adequately restrictiveenvironment in order to maintain neuronal cells at various stages ofdifferentiation, and can be employed, for instance, in cell culturesdesigned to test the specific activities of other trophic factors. Insuch embodiments of the subject method, the cultured cells can becontacted with a ptc-2 therapeutic, e.g., such as an agent identified inthe assays described above which potentiate ptc-2-dependent hedgehogbioactivities, in order to induce neuronal differentiation (e.g. of astem cell), or to maintain the integrity of a culture ofterminally-differentiated neuronal cells by preventing loss ofdifferentiation. Alternatively, a antagonist of hedgehog induction, ascertain of the ptc-2 homologs of the present invention are expected tobe, can be used to prevent differentiation of progenitor cells inculture.

[0157] To further illustrate uses of ptc-2 therapeutics which may beeither hedgehog agonists or antagonists, it is noted that intracerebralgrafting has emerged as an additional approach to central nervous systemtherapies. For example, one approach to repairing damaged brain tissuesinvolves the transplantation of cells from fetal or neonatal animalsinto the adult brain (Dunnett et al. (1987) J Exp Biol 123:265-289; andFreund et al. (1985) J Neurosci 5:603-616). Fetal neurons from a varietyof brain regions can be successfully incorporated into the adult brain,and such grafts can alleviate behavioral defects. For example, movementdisorder induced by lesions of dopaminergic projections to the basalganglia can be prevented by grafts of embryonic dopaminergic neurons.Complex cognitive functions that are impaired after lesions of theneocortex can also be partially restored by grafts of embryonic corticalcells. The differential use of hedgehog agonists and antagonists in theculture can control the timing and type of differentiation accessible bythe culture.

[0158] In addition to the implantation of cells cultured in the presenceof hedgehog agonists and antagonists and other in vitro uses, yetanother aspect of the present invention concerns the therapeuticapplication of a ptc-2 therapeutics to enhance survival of neurons andother neuronal cells in both the central nervous system and theperipheral nervous system. The ability of hedgehog protein to regulateneuronal differentiation during development of the nervous system andalso presumably in the adult state indicates that certain of thehedgehog proteins, and accordingly ptc-2 therapeutic which modulatehedgehog bioactivities, can be reasonably expected to facilitate controlof adult neurons with regard to maintenance, functional performance, andaging of normal cells; repair and regeneration processes in chemicallyor mechanically lesioned cells; and prevention of degeneration andpremature death which result from loss of differentiation in certainpathological conditions. In light of this understanding, the presentinvention specifically contemplates applications of the subject ptc-2therapeutics to the treatment of (prevention and/or reduction of theseverity of) neurological conditions deriving from: (i) acute, subacute,or chronic injury to the nervous system, including traumatic injury,chemical injury, vasal injury and deficits (such as the ischemiaresulting from stroke), together with infectious/inflammatory andtumor-induced injury; (ii) aging of the nervous system includingAlzheimer's disease; (iii) chronic neurodegenerative diseases of thenervous system, including Parkinson's disease, Huntington's chorea,amylotrophic lateral sclerosis and the like, as well as spinocerebellardegenerations; and (iv) chronic immunological diseases of the nervoussystem or affecting the nervous system, including multiple sclerosis.

[0159] Many neurological disorders are associated with degeneration ofdiscrete populations of neuronal elements and may be treatable with atherapeutic regimen which includes a ptc-2 therapeutic that acts as ahedgehog agonist. For example, Alzheimer's disease is associated withdeficits in several neurotransmitter systems, both those that project tothe neocortex and those that reside with the cortex. For instance, thenucleus basalis in patients with Alzheimer's disease have been observedto have a profound (75%) loss of neurons compared to age-matchedcontrols. Although Alzheimer's disease is by far the most common form ofdementia, several other disorders can produce dementia. Several of theseare degenerative diseases characterized by the death of neurons invarious parts of the central nervous system, especially the cerebralcortex. However, some forms of dementia are associated with degenerationof the thalamus or the white matter underlying the cerebral cortex.Here, the cognitive dysfunction results from the isolation of corticalareas by the degeneration of efferents and afferents. Huntington'sdisease involves the degeneration of intrastriatal and corticalcholinergic neurons and GABAergic neurons. Pick's disease is a severeneuronal degeneration in the neocortex of the frontal and anteriortemporal lobes, sometimes accompanied by death of neurons in thestriatum. Treatment of patients suffering from such degenerativeconditions can include the application of ptc-2 therapeutics in order tocontrol, for example, differentiation and apoptotic events which giverise to loss of neurons (e.g. to enhance survival of existing neurons)as well as promote differentiation and repopulation by progenitor cellsin the area affected.

[0160] In addition to degenerative-induced dementias, a pharmaceuticalpreparation of one or more of the subject ptc-2 therapeutics can beapplied opportunely in the treatment of neurodegenerative disorderswhich have manifestations of tremors and involuntary movements.Parkinson's disease, for example, primarily affects subcorticalstructures and is characterized by degeneration of the nigrostriatalpathway, raphe nuclei, locus cereleus, and the motor nucleus of vagus.Ballism is typically associated with damage to the subthalmic nucleus,often due to acute vascular accident. Also included are neurogenic andmyopathic diseases which ultimately affect the somatic division of theperipheral nervous system and are manifest as neuromuscular disorders.Examples include chronic atrophies such as amyotrophic lateralsclerosis, Guillain-Barre syndrome and chronic peripheral neuropathy, aswell as other diseases which can be manifest as progressive bulbarpalsies or spinal muscular atrophies. The present method is amenable tothe treatment of disorders of the cerebellum which result in hypotoniaor ataxia, such as those lesions in the cerebellum which producedisorders in the limbs ipsilateral to the lesion. For instance, apreparation of a ptc-2 therapeutic can used to treat a restricted formof cerebellar cortical degeneration involving the anterior lobes(verrnis and leg areas) such as is common in alcoholic patients.

[0161] In an illustrative embodiment, the subject method is used totreat amyotrophic lateral sclerosis. ALS is a name given to a complex ofdisorders that comprise upper and lower motor neurons. Patients maypresent with progressive spinal muscular atrophy, progressive bulbarpalsy, primary lateral sclerosis, or a combination of these conditions.The major pathological abnormality is characterized by a selective andprogressive degeneration of the lower motor neurons in the spinal cordand the upper motor neurons in the cerebral cortex. The therapeuticapplication of a hedgehog agonist can be used alone, or in conjunctionwith other neurotrophic factors such as CNTF, BDNF or NGF to preventand/or reverse motor neuron degeneration in ALS patients. ptc-2therapeutics of the present invention can also be used in the treatmentof autonomic disorders of the peripheral nervous system, which includedisorders affecting the innervation of smooth muscle and endocrinetissue (such as glandular tissue). For instance, the subject method canbe used to treat tachycardia or atrial cardiac arrythmias which mayarise from a degenerative condition of the nerves innervating thestriated muscle of the heart.

[0162] Furthermore, a potential role for certain of the ptc-2therapeutics derives from the role of hedgehog proteins in developmentand maintenance of dendritic processes of axonal neurons. Potentialroles for hedgehog agonists consequently include guidance for axonalprojections and the ability to promote differentiation and/ormaintenance of the innervating cells to their axonal processes.Accordingly, compositions comprising ptc-2 therapeutics which agonizehedgehog activity, may be employed to support the survival andreprojection of several types of ganglionic neurons sympathetic andsensory neurons as well as motor neurons. In particular, suchtherapeutic compositions may be useful in treatments designed to rescue,for example, various neurons from lesion-induced death as well asguiding reprojection of these neurons after such damage. Such diseasesinclude, but are not limited to, CNS trauma infarction, infection (suchas viral infection with varicella-zoster), metabolic disease,nutritional deficiency, toxic agents (such as cisplatin treatment).

[0163] Moreover, certain of the ptc-2 therapeutics (e.g., whichantagonize hedgehog induction) may be useful in the selective ablationof sensory neurons, for example, in the treatment of chronic painsyndromes.

[0164] As appropriate, ptc-2 therapeutics can be used in nerveprostheses for the repair of central and peripheral nerve damage. Inparticular, where a crushed or severed axon is intubulated by use of aprosthetic device, certain of ptc-2 therapeutics can be added to theprosthetic device to increase the rate of growth and regeneration of thedendritic processes. Exemplary nerve guidance channels are described inU.S. Pat. Nos. 5,092,871 and 4,955,892. Accordingly, a severed axonalprocess can be directed toward the nerve ending from which it wassevered by a prosthesis nerve guide.

[0165] In another embodiment, the subject method can be used in thetreatment of neoplastic or hyperplastic transformations such as mayoccur in the central nervous system. For instance, certain of the ptc-2therapeutics which induce differentiation of neuronal cells can beutilized to cause such transformed cells to become either post-mitoticor apoptotic. Treatment with a ptc-2 therapeutic may facilitatedisruption of autocrine loops, such as TGF-β or PDGF autostimulatoryloops, which are believed to be involved in the neoplastictransformation of several neuronal tumors. ptc-2 therapeutics may,therefore, thus be of use in the treatment of, for example, malignantgliomas, medulloblastomas, neuroectodermal tumors, and ependymonas.

[0166] Yet another aspect of the present invention concerns theapplication of the discovery that hedgehog proteins are morphogenicsignals involved in other vertebrate organogenic pathways in addition toneuronal differentiation as described above, having apparent roles inother endodermal patterning, as well as both mesodermal and endodermaldifferentiation processes. As described in the literature, Shh plays arole in proper limb growth and patterning by initiating expression ofsignaling molecules, including Bmp-2 in the mesoderm and Fgf-4 in theectoderm. Thus, it is contemplated by the invention that compositionscomprising certain of the ptc-2 therapeutics can also be utilized forboth cell culture and therapeutic methods involving generation andmaintenance of non-neuronal tissue.

[0167] In one embodiment, the present invention makes use of thediscovery that hedgehog proteins, such as Shh, are apparently involvedin controlling the development of stem cells responsible for formationof the digestive tract, liver, lungs, and other organs which derive fromthe primitive gut. Shh serves as an inductive signal from the endodermto the mesoderm, which is critical to gut morphogenesis. Therefore, forexample, hedgehog agonists can be employed in the development andmaintenance of an artificial liver which can have multiple metabolicfunctions of a normal liver. In an exemplary embodiment, a ptc-2therapeutic which acts as a hedgehog agonist can be used to inducedifferentiation of digestive tube stem cells to form hepatocyte cultureswhich can be used to populate extracellular matrices, or which can beencapsulated in biocompatible polymers, to form both implantable andextracorporeal artificial livers.

[0168] In another embodiment, therapeutic compositions of hedgehogagonists can be utilized in conjunction with transplantation of suchartificial livers, as well as embryonic liver structures, to promoteintraperitoneal implantation, vascularization, and in vivodifferentiation and maintenance of the engrafted liver tissue.

[0169] In yet another embodiment, ptc-2 therapeutics can be employedtherapeutically to regulate such organs after physical, chemical orpathological insult. For instance, therapeutic compositions comprisinghedgehog agonists can be utilized in liver repair subsequent to apartial hepatectomy. Similarly, therapeutic compositions containinghedgehog agonists can be used to promote regeneration of lung tissue inthe treatment of emphysema.

[0170] In still another embodiment of the present invention,compositions comprising ptc-2 therapeutics can be used in the in vitrogeneration of skeletal tissue, such as from skeletogenic stem cells, aswell as the in vivo treatment of skeletal tissue deficiencies. Thepresent invention particularly contemplates the use of ptc-2therapeutics which agonize a hedgehog a skeletogenic activity, such asan ability to induce chondrogenesis and/or osteogenesis. By “skeletaltissue deficiency”, it is meant a deficiency in bone or other skeletalconnective tissue at any site where it is desired to restore the bone orconnective tissue, no matter how the deficiency originated, e.g. whetheras a result of surgical intervention, removal of tumor, ulceration,implant, fracture, or other traumatic or degenerative conditions.

[0171] For instance, the present invention makes available effectivetherapeutic methods and compositions for restoring cartilage function toa connective tissue. Such methods are useful in, for example, the repairof defects or lesions in cartilage tissue which is the result ofdegenerative wear such as that which results in arthritis, as well asother mechanical derangements which may be caused by trauma to thetissue, such as a displacement of torn meniscus tissue, meniscectomy, alaxation of a joint by a torn ligament, malignment of joints, bonefracture, or by hereditary disease. The present reparative method isalso useful for remodeling cartilage matrix, such as in plastic orreconstructive surgery, as well as periodontal surgery. The presentmethod may also be applied to improving a previous reparative procedure,for example, following surgical repair of a meniscus, ligament, orcartilage. Furthermore, it may prevent the onset or exacerbation ofdegenerative disease if applied early enough after trauma.

[0172] In one embodiment of the present invention, the subject methodcomprises treating the afflicted connective tissue with atherapeutically sufficient amount of a hedgehog agonist, particularlyptc-2 therapeutic which agonizes Ihh activity, to generate a cartilagerepair response in the connective tissue by stimulating thedifferentiation and/or proliferation of chondrocytes embedded in thetissue. Induction of chondrocytes by treatment with a hedgehog agonistcan subsequently result in the synthesis of new cartilage matrix by thetreated cells. Such connective tissues as articular cartilage,interarticular cartilage (menisci), costal cartilage (connecting thetrue ribs and the sternum), ligaments, and tendons are particularlyamenable to treatment in reconstructive and/or regenerative therapiesusing the subject method. As used herein, regenerative therapies includetreatment of degenerative states which have progressed to the point ofwhich impairment of the tissue is obviously manifest, as well aspreventive treatments of tissue where degeneration is in its earlieststages or imminent. The subject method can further be used to preventthe spread of mineralisation into fibrotic tissue by maintaining aconstant production of new cartilage.

[0173] In an illustrative embodiment, the subject method can be used totreat cartilage of a diarthroidal joint, such as a knee, an ankle, anelbow, a ptc-2, a wrist, a knuckle of either a finger or toe, or atemperomandibular joint. The treatment can be directed to the meniscusof the joint, to the articular cartilage of the joint, or both. Tofurther illustrate, the subject method can be used to treat adegenerative disorder of a knee, such as which might be the result oftraumatic injury (e.g., a sports injury or excessive wear) orosteoarthritis. An injection of a ptc-2 therapeutic into the joint with,for instance, an arthroscopic needle, can be used to treat the afflictedcartilage. In some instances, the injected agent can be in the form of ahydrogel or other slow release vehicle described above in order topermit a more extended and regular contact of the agent with the treatedtissue.

[0174] The present invention further contemplates the use of the subjectmethod in the field of cartilage transplantation and prosthetic devicetherapies. To date, the growth of new cartilage from eithertransplantation of autologous or allogenic cartilage has been largelyunsuccessful. Problems arise, for instance, because the characteristicsof cartilage and fibrocartilage varies between different tissue: such asbetween articular, meniscal cartilage, ligaments, and tendons, betweenthe two ends of the same ligament or tendon, and between the superficialand deep parts of the tissue. The zonal arrangement of these tissues mayreflect a gradual change in mechanical properties, and failure occurswhen implanted tissue, which has not differentiated under thoseconditions, lacks the ability to appropriately respond. For instance,when meniscal cartilage is used to repair anterior cruciate ligaments,the tissue undergoes a metaplasia to pure fibrous tissue. By promotingchondrogenesis, the subject method can be used to particularly addressesthis problem, by causing the implanted cells to become more adaptive tothe new environment and effectively resemble hypertrophic chondrocytesof an earlier developmental stage of the tissue. Thus, the action ofchondrogenesis in the implanted tissue, as provided by the subjectmethod, and the mechanical forces on the actively remodeling tissue cansynergize to produce an improved implant more suitable for the newfunction to which it is to be put.

[0175] In similar fashion, the subject method can be applied toenhancing both the generation of prosthetic cartilage devices and totheir implantation. The need for improved treatment has motivatedresearch aimed at creating new cartilage that is based oncollagen-glycosaminoglycan templates (Stone et al. (1990) Clin OrthopRelat Red 252:129), isolated chondrocytes (Grande et al. (1989) J OrthopRes 7:208; and Takigawa et al. (1987) Bone Miner 2:449), andchondrocytes attached to natural or synthetic polymers (Walitani et al.(1989) J Bone Jt Surg 71 B:74; Vacanti et al. (1991) Plast Reconstr Surg88:753; von Schroeder et al. (1991) J Biomed Mater Res 25:329; Freed etal. (1993) J Biomed Mater Res 27:11; and the Vacanti et al. U.S. Pat.No. 5,041,138). For example, chondrocytes can be grown in culture onbiodegradable, biocompatible highly porous scaffolds formed frompolymers such as polyglycolic acid, polylactic acid, agarose gel, orother polymers which degrade over time as function of hydrolysis of thepolymer backbone into innocuous monomers. The matrices are designed toallow adequate nutrient and gas exchange to the cells until engraftmentoccurs. The cells can be cultured in vitro until adequate cell volumeand density has developed for the cells to be implanted. One advantageof the matrices is that they can be cast or molded into a desired shapeon an individual basis, so that the final product closely resembles thepatient's own ear or nose (by way of example), or flexible matrices canbe used which allow for manipulation at the time of implantation, as ina joint.

[0176] In one embodiment of the subject method, the implants arecontacted with a ptc-2 therapeutic during the culturing process, such asan Ihh agonist, in order to induce and/or maintain differentiatedchondrocytes in the culture in order as to further stimulate cartilagematrix production within the implant. In such a manner, the culturedcells can be caused to maintain a phenotype typical of a chondrogeniccell (i.e. hypertrophic), and hence continue the population of thematrix and production of cartilage tissue.

[0177] In another embodiment, the implanted device is treated with aptc-2 therapeutic in order to actively remodel the implanted matrix andto make it more suitable for its intended function. As set out abovewith respect to tissue transplants, the artificial transplants sufferfrom the same deficiency of not being derived in a setting which iscomparable to the actual mechanical environment in which the matrix isimplanted. The activation of the chondrocytes in the matrix by thesubject method can allow the implant to acquire characteristics similarto the tissue for which it is intended to replace.

[0178] In yet another embodiment, the subject method is used to enhanceattachment of prosthetic devices. To illustrate, the subject method canbe used in the implantation of a periodontal prosthesis, wherein thetreatment of the surrounding connective tissue stimulates formation ofperiodontal ligament about the prosthesis, as well as inhibits formationof fibrotic tissue proximate the prosthetic device.

[0179] In still further embodiments, the subject method can be employedfor the generation of bone (osteogenesis) at a site in the animal wheresuch skeletal tissue is deficient. Indian hedgehog is particularlyassociated with the hypertrophic chondrocytes that are ultimatelyreplaced by osteoblasts. For instance, administration of a ptc-2therapeutic of the present invention can be employed as part of a methodfor treating bone loss in a subject, e.g. to prevent and/or reverseosteoporosis and other osteopenic disorders, as well as to regulate bonegrowth and maturation. For example, preparations comprising hedgehogagonists can be employed, for example, to induce endochondralossification, at least so far as to facilitate the formation ofcartilaginous tissue precursors to form the “model” for ossification.Therapeutic compositions of ptc-² therapeutics can be supplemented, ifrequired, with other osteoinductive factors, such as bone growth factors(e.g. TGF-β factors, such as the bone morphogenetic factors BMP-2 andBMP-4, as well as activin), and may also include, or be administered incombination with, an inhibitor of bone resorption such as estrogen,bisphosphonate, sodium fluoride, calcitonin, or tamoxifen, or relatedcompounds. However, it will be appreciated that hedgehog proteins, suchas Ihh and Shh are likely to be upstream of BMPs, e.g. treatment. with ahedgehog agonist will have the advantage of initiating endogenousexpression of BMPs along with other factors.

[0180] In yet another embodiment, the ptc-2 therapeutic of the presentinvention can be used in the treatment of testicular cells, so as tomodulate spermatogenesis. In light of the finding that hedgehog proteinsare involved in the differentiation and/or proliferation and maintenanceof testicular germ cells, hedgehog antagonist can be utilized to blockthe action of a naturally-occurring hedgehog protein. In a preferredembodiment, the ptc-2 therapeutic inhibits the biological activity ofDhh with respect to spermatogenesis, by competitively binding hedgehogin the testis. That is, the ptc-2 therapeutic can be administered as acontraceptive formulation. Alternatively, ptc-2 therapeutics whichagonize the spermatogenic activity of Dhh can be used as fertilityenhancers. In similar fashion, hedgehog agonists and antagonists arepotentially useful for modulating normal ovarian function.

[0181] Another aspect of the invention features transgenic non-humananimals which express a heterologous ptc-2 gene of the presentinvention, and/or which have had one or more genomic ptc-2 genesdisrupted in at least a tissue or cell-types of the animal. Accordingly,the invention features an animal model for developmental diseases, whichanimal has one or more ptc-2 allele which is mis-expressed. For example,an animal can be generated which has one or more ptc-2 alleles deletedor otherwise rendered inactive. Such a model can then be used to studydisorders arising from mis-expressed ptc-2 genes, as well as forevaluating potential therapies for similar disorders.

[0182] The transgenic animals of the present invention all includewithin a plurality of their cells a transgene of the present invention,which transgene alters the phenotype of the “host cell” with respect toregulation by the ptc-2 protein, e.g., of cell growth, death and/ordifferentiation. Since it is possible to produce transgenic organisms ofthe invention utilizing one or more of the transgene constructsdescribed herein, a general description will be given of the productionof transgenic organisms by referring generally to exogenous geneticmaterial. This general description can be adapted by those skilled inthe art in order to incorporate specific transgene sequences intoorganisms utilizing the methods and materials described herein and thosegenerally known in the art.

[0183] In one embodiment, the transgene construct is a knockoutconstruct. Such transgene constructs usually are insertion-type orreplacement-type constructs (Hasty et al. (1991) Mol Cell Biol 11:4509).The transgene constructs for disruption of a ptc-2 gene are designed tofacilitate homologous recombination with a portion of the genomic ptc-2gene so as to prevent the functional expression of the endogenous ptc-2gene. In preferred embodiments, the nucleotide sequence used as theknockout construct can be comprised of (1) DNA from some portion of theendogenous ptc-2 gene (exon sequence, intron sequence, promotersequences, etc.) which direct recombination and (2) a marker sequencewhich is used to detect the presence of the knockout construct in thecell. The knockout construct is inserted into a cell, and integrateswith the genomic DNA of the cell in such a position so as to prevent orinterrupt transcription of the native ptc-2 gene. Such insertion canoccur by homologous recombination, i.e., regions of the knockoutconstruct that are homologous to the endogenous ptc-2 gene sequencehybridize to the genomic DNA and recombine with the genomic sequences sothat the construct is incorporated into the corresponding position ofthe genomic DNA. The knockout construct can comprise (1) a full orpartial sequence of one or more exons and/or introns of the ptc-2 geneto be disrupted, (2) sequences which flank the 5′ and 3′ ends of thecoding sequence of the ptc-2 gene, or (3) a combination thereof.

[0184] A preferred knockout construct will delete, by targetedhomologous recombination, essential structural elements of an endogenousptc-2 gene. For example, the targeting construct can recombine with thegenomic ptc-2 gene can delete a portion of the coding sequence, and/oressential transcriptional regulatory sequences of the gene.

[0185] Alternatively, the knockout construct can be used to interruptessential structural and/or regulatory elements of an endogenous ptc-2gene by targeted insertion of a polynucleotide sequence. For instance, aknockout construct can recombine with a ptc-2 gene and insert anonhomologous sequence, such as a neo expression cassette, into astructural element (e.g., an exon) and/or regulatory element (e.g.,enhancer, promoter, intron splice site, polyadenylation site, etc.) toyield a targeted ptc-2 allele having an insertional disruption. Theinserted nucleic acid can range in size from 1 nucleotide (e.g., toproduce a frameshift) to several kilobases or more, and is limited onlyby the efficiency of the targeting technique.

[0186] Depending of the location and characteristics of the disruption,the transgene construct can be used to generate a transgenic animal inwhich substantially all expression of the targeted ptc-2 gene isinhibited in at least a portion of the animal's cells. If onlyregulatory elements are targeted, some low-level expression of thetargeted gene may occur (i.e., the targeted allele is “leaky”).

[0187] The nucleotide sequence(s) comprising the knockout construct(s)can be obtained using methods well known in the art. Such methodsinclude, for example, screening genomic libraries with ptc-2 cDNA probesin order to identify the corresponding genomic ptc-2 gene and regulatorysequences. Alternatively, where the cDNA sequence is to be used as partof the knockout construct, the cDNA may be obtained by screening a cDNAlibrary as set out above.

[0188] In another embodiment, the “transgenic non-human animals” of theinvention are produced by introducing transgenes into the germline ofthe non-human animal. Embryonal target cells at various developmentalstages can be used to introduce transgenes. Different methods are useddepending on the stage of development of the embryonal target cell. Thespecific line(s) of any animal used to practice this invention areselected for general good health, good embryo yields, good pronuclearvisibility in the embryo,and good reproductive fitness. In addition, thehaplotype is a significant factor. For example, when transgenic mice areto be produced, strains such as C57BL/6 or FVB lines are often used(Jackson Laboratory, Bar Harbor, Me.). Preferred strains are those withH-2^(b), H-2^(d) or H-2^(q) haplotypes such as C57BL/6 or DBA/1. Theline(s) used to practice this invention may themselves be transgenics,and/or may be knockouts (i.e., obtained from animals which have one ormore genes partially or completely suppressed).

[0189] In one embodiment, the transgene construct is introduced into asingle stage embryo. The zygote is the best target for micro-injection.The use of zygotes as a target for gene transfer has a major advantagein that in most cases the injected DNA will be incorporated into thehost gene before the first cleavage (Brinster et al. (1985) PNAS82:4438-4442). As a consequence, all cells of the transgenic animal willcarry the incorporated transgene. This will in general also be reflectedin the efficient transmission of the transgene to offspring of thefounder since 50% of the germ cells will harbor the transgene.

[0190] Introduction of the transgene nucleotide sequence into the embryomay be accomplished by any means known in the art such as, for example,microinjection, electroporation, or lipofection. Following introductionof the transgene nucleotide sequence into the embryo, the embryo may beincubated in vitro for varying amounts of time, or reimplanted into thesurrogate host, or both. In vitro incubation to maturity is within thescope of this invention. One common method in to incubate the embryos invitro for about 1-7 days, depending on the species, and then reimplantthem into the surrogate host.

[0191] Any technique which allows for the addition of the exogenousgenetic material into nucleic genetic material can be utilized so longas it is not destructive to the cell, nuclear membrane or other existingcellular or genetic structures. The exogenous genetic material ispreferentially inserted into the nucleic genetic material bymicroinjection. Microinjection of cells and cellular structures is knownand is used in the art.

[0192] Reimplantation is accomplished using standard methods. Usually,the surrogate host is anesthetized, and the embryos are inserted intothe oviduct. The number of embryos implanted into a particular host willvary by species, but will usually be comparable to the number of offspring the species naturally produces.

[0193] Transgenic offspring of the surrogate host may be screened forthe presence and/or expression of the transgene by any suitable method.Screening is often accomplished by Southern blot or Northern blotanalysis, using a probe that is complementary to at least a portion ofthe transgene. Western blot analysis using an antibody against theprotein encoded by the transgene may be employed as an alternative oradditional method for screening for the presence of the transgeneproduct. Typically, DNA is prepared from excised tissue and analyzed bySouthern analysis or PCR for the transgene. Alternatively, the tissuesor cells believed to express the transgene at the highest levels aretested for the presence and expression of the transgene using Southernanalysis or PCR, although any tissues or cell types may be used for thisanalysis.

[0194] Retroviral infection can also be used to introduce transgene intoa non-human animal. The developing non-human embryo can be cultured invitro to the blastocyst stage. During this time, the blastomeres can betargets for retroviral infection (Jaenich, R. (1976) PNAS 73:1260-1264).Efficient infection of the blastomeres is obtained by enzymatictreatment to remove the zona pellucida (Manipulating the Mouse Embryo,Hogan eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,1986). The viral vector system used to introduce the transgene istypically a replication-defective retrovirus carrying the transgene(Jahner et al. (1985) PNAS 82:6927-6931; Van der Putten et al. (1985)PNAS 82:6148-6152). Transfection is easily and efficiently obtained byculturing the blastomeres on a monolayer of virus-producing cells (Vander Putten, supra; Stewart et al. (1987) EMBO J. 6:383-388).Alternatively, infection can be performed at a later stage. Virus orvirus-producing cells can be injected into the blastocoele (Jahner etal. (1982) Nature 298:623-628). Most of the founders will be mosaic forthe transgene since incorporation occurs only in a subset of the cellswhich formed the transgenic non-human animal. Further, the founder maycontain various retroviral insertions of the transgene at differentpositions in the genome which generally will segregate in the offspring.In addition, it is also possible to introduce transgenes into the germline by intrauterine retroviral infection of the midgestation embryo(Jahner et al. (1982) supra).

[0195] A third type of target cell for transgene introduction is theembryonal stem cell (ES). ES cells are obtained from pre-implantationembryos cultured in vitro and fused with embryos (Evans et al. (1981)Nature 292:154-156; Bradley et al. (1984) Nature 309:255-258; Gossler etal. (1986 PNAS 83: 9065-9069; and Robertson et al. (1986) Nature322:445-448). Transgenes can be efficiently introduced into the ES cellsby DNA transfection or by retrovirus-mediated transduction. Suchtransformed ES cells can thereafter be combined with blastocysts from anon-human animal. The ES cells thereafter colonize the embryo andcontribute to the germ line of the resulting chimeric animal. For reviewsee Jaenisch, R. (1988) Science 240:1468-1474.

[0196] In one embodiment, gene targeting, which is a method of usinghomologous recombination to modify an animal's genome, can be used tointroduce changes into cultured embryonic stem cells. By targeting theptc-2 gene in ES cells, these changes can be introduced into thegermlines of animals to generate chimeras. The gene targeting procedureis accomplished by introducing into tissue culture cells a DNA targetingconstruct that includes a segment homologous to a ptc-2 locus, and whichalso includes an intended sequence modification to the ptc-2 genomicsequence (e.g., insertion, deletion, point mutation). The treated cellsare then screened for accurate targeting to identify and isolate thosewhich have been properly targeted.

[0197] Gene targeting in embryonic stem cells is in fact a schemecontemplated by the present invention as a means for disrupting a ptc-2gene function through the use of a targeting transgene constructdesigned to undergo homologous recombination with ptc-2 genomicsequences. Targeting construct can be arranged so that, uponrecombination with an element of a ptc-2 gene, a positive selectionmarker is inserted into (or replaces) coding sequences of the targetedptc-2 gene. The inserted sequence functionally disrupts the ptc-2 gene,while also providing a positive selection trait.

[0198] Generally, the embryonic stem cells (ES cells) used to producethe knockout animals will be of the same species as the knockout animalto be generated. Thus for example, mouse embryonic stem cells willusually be used for generation of a ptc-2-knockout mice.

[0199] Embryonic stem cells are generated and maintained using methodswell known to the skilled artisan such as those described by Doetschmanet al. (1985) J. Embryol. Exp. Morphol. 87:27-45). Any line of ES cellscan be used, however, the line chosen is typically selected for theability of the cells to integrate into and become part of the germ lineof a developing embryo so as to create germ line transmission of theknockout construct. Thus, any ES cell line that is believed to have thiscapability is suitable for use herein. The cells are cultured andprepared for knockout construct insertion using methods well known tothe skilled artisan, such as those set forth by Robertson in:Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J.Robertson, ed. IRL Press, Washington, D.C. [1987]); by Bradley et al.(1986) Current Topics in Devel. Biol. 20:357-371); and by Hogan et al.(Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. [1986]).

[0200] Insertion of the knockout construct into the ES cells can beaccomplished using a variety of methods well known in the art includingfor example, electroporation, microinjection, and calcium phosphatetreatment. A preferred method of insertion is electroporation.

[0201] Each knockout construct to be inserted into the cell must firstbe in the linear form. Therefore, if the knockout construct has beeninserted into a vector, linearization is accomplished by digesting theDNA with a suitable restriction endonuclease selected to cut only withinthe vector sequence and not within the knockout construct sequence.

[0202] For insertion, the knockout construct is added to the ES cellsunder appropriate conditions for the insertion method chosen, as isknown to the skilled artisan. Where more than one construct is to beintroduced into the ES cell, each knockout construct can be introducedsimultaneously or one at a time.

[0203] If the ES cells are to be electroporated, the ES cells andknockout construct DNA are exposed to an electric pulse using anelectroporation machine and following the manufacturer's guidelines foruse. After electroporation, the ES cells are typically allowed torecover under suitable incubation conditions. The cells are thenscreened for the presence of the knockout construct.

[0204] Screening can be accomplished using a variety of methods. Wherethe marker gene is an antibiotic resistance gene, the ES cells may becultured in the presence of an otherwise lethal concentration ofantibiotic. Those ES cells that survive have presumably integrated theknockout construct. If the marker gene is other than an antibioticresistance gene, a Southern blot of the ES cell genomic DNA can beprobed with a sequence of DNA designed to hybridize only to the markersequence Alternatively, PCR can be used. Finally, if the marker gene isa gene that encodes an enzyme whose activity can be detected (e.g.,β-galactosidase), the enzyme substrate can be added to the cells undersuitable conditions, and the enzymatic activity can be analyzed. Oneskilled in the art will be familiar with other useful markers and themeans for detecting their presence in a given cell. All such markers arecontemplated as being included within the scope of the teaching of thisinvention.

[0205] The knockout construct may integrate into several locations inthe ES cell genome, and may integrate into a different location in eachES cell's genome due to the occurrence of random insertion events. Thedesired location of insertion is in a complementary position to the DNAsequence to be knocked out, e.g., the ptc-2 coding sequence,transcriptional regulatory sequence, etc. Typically, less than about 1-5percent of the ES cells that take up the knockout construct willactually integrate the knockout construct in the desired location. Toidentify those ES cells with proper integration of the knockoutconstruct, total DNA can be extracted from the ES cells using standardmethods. The DNA can then be probed on a Southern blot with a probe orprobes designed to hybridize in a specific pattern to genomic DNAdigested with particular restriction enzyme(s). Alternatively, oradditionally, the genomic DNA can be amplified by PCR with probesspecifically designed to amplify DNA fragments of a particular size andsequence (i.e., only those cells containing the knockout construct inthe proper position will generate DNA fragments of the proper size).

[0206] After suitable ES cells containing the knockout construct in theproper location have been identified, the cells can be inserted into anembryo. Insertion may be accomplished in a variety of ways known to theskilled artisan, however a preferred method is by microinjection. Formicroinjection, about 10-30 cells are collected into a micropipet andinjected into embryos that are at the proper stage of development topermit integration of the foreign ES cell containing the knockoutconstruct into the developing embryo. For instance, the transformed EScells can be microinjected into blastocytes.

[0207] After the ES cell has been introduced into the embryo, the embryomay be implanted into the uterus of a pseudopregnant foster mother forgestation. While any foster mother may be used, the foster mother istypically selected for her ability to breed and reproduce well, and forher ability to care for the young. Such foster mothers are typicallyprepared by mating with vasectomized males of the same species. Thestage of the pseudopregnant foster mother is important for successfulimplantation, and it is species dependent.

[0208] Offspring that are born to the foster mother may be screenedinitially for ptc-2 disruptants, DNA from tissue of the offspring may bescreened for the presence of the knockout construct using Southern blotsand/or PCR as described above. Offspring that appear to be mosaics maythen be crossed to each other, if they are believed to carry theknockout construct in their germ line, in order to generate homozygousknockout animals. Homozygotes may be identified by Southern blotting ofequivalent amounts of genomic DNA from animals that are the product ofthis cross, as well as animals that are known heterozygotes and wildtype animals.

[0209] Other means of identifying and characterizing the knockoutoffspring are available. For example, Northern blots can be used toprobe the mRNA for the presence or absence of transcripts of either theptc-2 gene, the marker gene, or both. In addition, Western blots can beused to assess the (loss of) level of expression of the ptc-2 geneknocked out in various tissues of the offspring by probing the Westernblot with an antibody against the ptc-2 protein, or an antibody againstthe marker gene product, where this gene is expressed. Finally, in situanalysis (such as fixing the cells and labeling with antibody) and/orFACS (fluorescence activated cell sorting) analysis of various cellsfrom the offspring can be conducted using suitable antibodies or ptc-2ligands, e.g., hedgehog proteins, to look for the presence or absence ofthe knockout construct gene product.

[0210] Animals containing more than one knockout construct and/or morethan one transgene expression construct are prepared in any of severalways. The preferred manner of preparation is to generate a series ofanimals, each containing a desired transgenic phenotypes. Such animalsare bred together through a series of crosses, backcrosses andselections, to ultimately generate a single animal containing alldesired knockout constructs and/or expression constructs, where theanimal is otherwise congenic (genetically identical) to the wild typeexcept for the presence of the knockout construct(s) and/ortransgene(s). Thus, a transgenic avian species can be generated bybreeding a first transgenic bird in which the wild-type ptc-2 gene isdisrupted with a second transgenic bird which has been engineered toexpress a mutant ptc-2 which retains most other biological functions ofthe receptor.

[0211] The transformed animals, their progeny, and cell lines of thepresent invention provide several important uses that will be readilyapparent to one of ordinary skill in the art.

[0212] To illustrate, the transgenic animals and cell lines areparticularly useful in screening compounds that have potential asprophylactic or therapeutic treatments of diseases such as may involveaberrant expression, or loss, of a ptc-2 gene, or aberrant or unwantedactivation of receptor signaling. Screening for a useful drug wouldinvolve administering the candidate drug over a range of doses to thetransgenic animal, and assaying at various time points for the effect(s)of the drug on the disease or disorder being evaluated. Alternatively,or additionally, the drug could be administered prior to orsimultaneously with exposure to induction of the disease, if applicable.

[0213] In one embodiment, candidate compounds are screened by beingadministered to the transgenic animal, over a range of doses, andevaluating the animal's physiological response to the compound(s) overtime. Administration may be oral, or by suitable injection, depending onthe chemical nature of the compound being evaluated. In some cases, itmay be appropriate to administer the compound in conjunction withco-factors that would enhance the efficacy of the compound.

[0214] In screening cell lines derived from the subject transgenicanimals for compounds useful in treating various disorders, the testcompound is added to the cell culture medium at the appropriate time,and the cellular response to the compound is evaluated over time usingthe appropriate biochemical and/or histological assays. In some cases,it may be appropriate to apply the compound of interest to the culturemedium in conjunction with co-factors that would enhance the efficacy ofthe compound.

EXAMPLE 1 Cloning of Human ptc-2

[0215] cDNA for PCR was made from ClonTech human brain mRNA primed withrandom hexamers. Four PCR primers were made based on alignment analysisof ptc-1 amino acid sequences from different species (human, mouse,chick, zebrafish, and Drosophila) and the partial mouse ptc-2 sequence(T. Takabatake, et al., (1997) FEBS Letters 410:485): P106,CAAACTCCAAGGGGGCTCTG; P107, CACAAAGCCCAAGACCTGAG; P143,TGGAATTCTTGGGTNGTNGC; P144, GAYTGYTTYTGGGARGG. Primers P143 and P144were first used to enrich for ptc-2 DNA using the following PCR profile:95° C. for 20 sec, 52° for 15 sec, and 72° C. for 30 sec. Then the PCRmixture was diluted 1:20 and used as template for nested PCR with P 106and P 107 as primers. The correct-sized band was excised, cloned, andsequenced.

[0216] PCR primers were designed based on the resulting partial humanptc-2 sequence to perform RACE (Rapid Amplification of cDNA Ends) inboth directions. The complete 5′ coding region was identified with RACE,and about 700 bp of cDNA sequence was obtained on the 3′ side. Two PCRprimers were designed based on this 3′ ptc-2 sequence, and used toscreen a human amygdala cDNA library. A 2.5 kb clone was selected andsequenced.

[0217] All the 5′ RACE sequence was either confirmed by an independentPCR fragment, or by a cDNA clone from a random-primed human fetal braincDNA library (ClonTech).

[0218] The complete cDNA was assembled with the 2.5 kb cDNA clone, a 1.1kb cDNA clone from the human fetal brain cDNA library, and a 1.7 kb PCRfragment which was sequenced completely.

[0219] All of the above-cited references and publications are herebyincorporated by reference.

Equivalents

[0220] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, numerous equivalents to thespecific polypeptides, nucleic acids, methods, assays and reagentsdescribed herein. Such equivalents are considered to be within the scopeof this invention.

1 6 1 4391 DNA Homo sapiens CDS (297)..(3905) 1 tgcgtctcgg gccatcaattccgcctgcgc gcgggaccca ttcctcctcc agccgccacg 60 gactacggcc cacggagcgggtgaatcccg gcgccgcgcc ccggacccgc agctccctgc 120 actcctccct cccagccgctttaacaccca caccccacag tctctcccac gcccgcgcct 180 tggcggcccc actgaatccctacgcggggc ccagcggtac cgggagaccg ggctagccta 240 tgggagcgcc cagataacgcgggttggggg cgcccgcgcc ccccatcccc gccagc atg 299 Met 1 act cga tcg ccgccc ctc aga gag ctg ccc ccg agt tac aca ccc cca 347 Thr Arg Ser Pro ProLeu Arg Glu Leu Pro Pro Ser Tyr Thr Pro Pro 5 10 15 gct cga acc gca gcaccc cag atc cta gct ggg agc ctg aag gct cca 395 Ala Arg Thr Ala Ala ProGln Ile Leu Ala Gly Ser Leu Lys Ala Pro 20 25 30 ctc tgg ctt cgt gct tacttc cag ggc ctg ctc ttc tct ctg gga tgc 443 Leu Trp Leu Arg Ala Tyr PheGln Gly Leu Leu Phe Ser Leu Gly Cys 35 40 45 ggg atc cag aga cat tgt ggcaaa gtg ctc ttt ctg gga ctg ttg gcc 491 Gly Ile Gln Arg His Cys Gly LysVal Leu Phe Leu Gly Leu Leu Ala 50 55 60 65 ttt ggg gcc ctg gca tta ggtctc cgc atg gcc att att gag aca aac 539 Phe Gly Ala Leu Ala Leu Gly LeuArg Met Ala Ile Ile Glu Thr Asn 70 75 80 ttg gaa cag ctc tgg gta gaa gtgggc agc cgg gtg agc cag gag ctg 587 Leu Glu Gln Leu Trp Val Glu Val GlySer Arg Val Ser Gln Glu Leu 85 90 95 cat tac acc aag gag aag ctg ggg gaggag gct gca tac acc tct cag 635 His Tyr Thr Lys Glu Lys Leu Gly Glu GluAla Ala Tyr Thr Ser Gln 100 105 110 atg ctg ata cag acc gca cgc cag gaggga gag aac atc ctc aca ccc 683 Met Leu Ile Gln Thr Ala Arg Gln Glu GlyGlu Asn Ile Leu Thr Pro 115 120 125 gaa gca ctt ggc ctc cac ctc cag gcagcc ctc act gcc agt aaa gtc 731 Glu Ala Leu Gly Leu His Leu Gln Ala AlaLeu Thr Ala Ser Lys Val 130 135 140 145 caa gta tca ctc tat ggg aag tcctgg gat ttg aac aaa atc tgc tac 779 Gln Val Ser Leu Tyr Gly Lys Ser TrpAsp Leu Asn Lys Ile Cys Tyr 150 155 160 aag tca gga gtt ccc ctt att gaaaat gga atg att gag cgg atg att 827 Lys Ser Gly Val Pro Leu Ile Glu AsnGly Met Ile Glu Arg Met Ile 165 170 175 gag aag ctg ttt ccg tgc gtg atcctc acc ccc ctc gac tgc ttc tgg 875 Glu Lys Leu Phe Pro Cys Val Ile LeuThr Pro Leu Asp Cys Phe Trp 180 185 190 gag gga gcc aaa ctc caa ggg ggctcc gcc tac ctg ccc ggc cgc ccg 923 Glu Gly Ala Lys Leu Gln Gly Gly SerAla Tyr Leu Pro Gly Arg Pro 195 200 205 gat atc cag tgg acc aac ctg gatcca gag cag ctg ctg gag gag ctg 971 Asp Ile Gln Trp Thr Asn Leu Asp ProGlu Gln Leu Leu Glu Glu Leu 210 215 220 225 ggt ccc ttt gcc tcc ctt gagggc ttc cgg gag ctg cta gac aag gca 1019 Gly Pro Phe Ala Ser Leu Glu GlyPhe Arg Glu Leu Leu Asp Lys Ala 230 235 240 cag gtg ggc cag gcc tac gtgggg cgg ccc tgt ctg cac cct gat gac 1067 Gln Val Gly Gln Ala Tyr Val GlyArg Pro Cys Leu His Pro Asp Asp 245 250 255 ctc cac tgc cca cct agt gccccc aac cat cac agc agg cag gct ccc 1115 Leu His Cys Pro Pro Ser Ala ProAsn His His Ser Arg Gln Ala Pro 260 265 270 aat gtg gct cac gag ctg agtggg ggc tgc cat ggc ttc tcc cac aaa 1163 Asn Val Ala His Glu Leu Ser GlyGly Cys His Gly Phe Ser His Lys 275 280 285 ttc atg cac tgg cag gag gaattg ctg ctg gga ggc atg gcc aga gac 1211 Phe Met His Trp Gln Glu Glu LeuLeu Leu Gly Gly Met Ala Arg Asp 290 295 300 305 ccc caa gga gag ctg ctgagg gca gag gcc ctg cag agc acc ttc ttg 1259 Pro Gln Gly Glu Leu Leu ArgAla Glu Ala Leu Gln Ser Thr Phe Leu 310 315 320 ctg atg agt ccc cgc cagctg tac gag cat ttc cgg ggt gac tat cag 1307 Leu Met Ser Pro Arg Gln LeuTyr Glu His Phe Arg Gly Asp Tyr Gln 325 330 335 aca cat gac att ggc tggagt gag gag cag gcc agc aca gtg cta caa 1355 Thr His Asp Ile Gly Trp SerGlu Glu Gln Ala Ser Thr Val Leu Gln 340 345 350 gcc tgg cag cgg cgc tttgtg cag ctg gcc cag gag gcc ctg cct gag 1403 Ala Trp Gln Arg Arg Phe ValGln Leu Ala Gln Glu Ala Leu Pro Glu 355 360 365 aac gct tcc cag cag atccat gcc ttc tcc tcc acc acc ctg gat gac 1451 Asn Ala Ser Gln Gln Ile HisAla Phe Ser Ser Thr Thr Leu Asp Asp 370 375 380 385 atc ctg cat gcg ttctct gaa gtc agt gct gcc cgt gtg gtg gga ggc 1499 Ile Leu His Ala Phe SerGlu Val Ser Ala Ala Arg Val Val Gly Gly 390 395 400 tat ctg ctc atg ctggcc tat gcc tgt gtg acc atg ctg cgg tgg gac 1547 Tyr Leu Leu Met Leu AlaTyr Ala Cys Val Thr Met Leu Arg Trp Asp 405 410 415 tgc gcc cag tcc cagggt tcc gtg ggc ctt gcc ggg gta ctg ctg gtg 1595 Cys Ala Gln Ser Gln GlySer Val Gly Leu Ala Gly Val Leu Leu Val 420 425 430 gcc ctg gcg gtg gcctca ggc ctt ggg ctc tgt gcc ctg ctc ggc atc 1643 Ala Leu Ala Val Ala SerGly Leu Gly Leu Cys Ala Leu Leu Gly Ile 435 440 445 acc ttc aat gct gccact acc cag gtg ctg ccc ttc ttg gct ctg gga 1691 Thr Phe Asn Ala Ala ThrThr Gln Val Leu Pro Phe Leu Ala Leu Gly 450 455 460 465 atc ggc gtg gatgac gta ttc ctg ctg gcg cat gcc ttc aca gag gct 1739 Ile Gly Val Asp AspVal Phe Leu Leu Ala His Ala Phe Thr Glu Ala 470 475 480 ctg cct ggc acccct ctc cag gag cgc atg ggc gag tgt ctg cag cgc 1787 Leu Pro Gly Thr ProLeu Gln Glu Arg Met Gly Glu Cys Leu Gln Arg 485 490 495 acg ggc acc agtgtc gta ctc aca tcc atc aac aac atg gcc gcc ttc 1835 Thr Gly Thr Ser ValVal Leu Thr Ser Ile Asn Asn Met Ala Ala Phe 500 505 510 ctc atg gct gccctc gtt ccc atc cct gcg ctg cga gcc ttc tcc cta 1883 Leu Met Ala Ala LeuVal Pro Ile Pro Ala Leu Arg Ala Phe Ser Leu 515 520 525 cag gcg gcc atagtg gtt ggc tgc acc ttt gta gcc gtg atg ctt gtc 1931 Gln Ala Ala Ile ValVal Gly Cys Thr Phe Val Ala Val Met Leu Val 530 535 540 545 ttc cca gccatc ttc agc ttg gac tta cgg cgg cgc cac tgc cag cgc 1979 Phe Pro Ala IlePhe Ser Leu Asp Leu Arg Arg Arg His Cys Gln Arg 550 555 560 ctt gat gtgctc tgc tgc ttc tcc agt ccc tgc tct gct cag gtg att 2027 Leu Asp Val LeuCys Cys Phe Ser Ser Pro Cys Ser Ala Gln Val Ile 565 570 575 cag atc ctgccc cag gag ctg ggg gac ggg aca gta cca gtg ggc att 2075 Gln Ile Leu ProGln Glu Leu Gly Asp Gly Thr Val Pro Val Gly Ile 580 585 590 gcc cac ctcact gcc aca gtt caa gcc ttt acc cac tgt gag gcc agc 2123 Ala His Leu ThrAla Thr Val Gln Ala Phe Thr His Cys Glu Ala Ser 595 600 605 agc cag catgtg gtc acc atc ctg cct ccc caa gcc cac ctg gtg ccc 2171 Ser Gln His ValVal Thr Ile Leu Pro Pro Gln Ala His Leu Val Pro 610 615 620 625 cca ccttct gac cca ctg ggc tct gag ctc ttc agc cct gga ggg tcc 2219 Pro Pro SerAsp Pro Leu Gly Ser Glu Leu Phe Ser Pro Gly Gly Ser 630 635 640 aca cgggac ctt cta ggc cag gag gag gag aca agg cag aag gca gcc 2267 Thr Arg AspLeu Leu Gly Gln Glu Glu Glu Thr Arg Gln Lys Ala Ala 645 650 655 tgc aagtcc ctg ccc tgt gcc cgc tgg aat ctt gcc cat ttc gcc cgc 2315 Cys Lys SerLeu Pro Cys Ala Arg Trp Asn Leu Ala His Phe Ala Arg 660 665 670 tat cagttt gcc ccg ttg ctg ctc cag tca cat gcc aag gcc atc gtg 2363 Tyr Gln PheAla Pro Leu Leu Leu Gln Ser His Ala Lys Ala Ile Val 675 680 685 ctg gtgctc ttt ggt gct ctt ctg ggc ctg agc ctc tac gga gcc acc 2411 Leu Val LeuPhe Gly Ala Leu Leu Gly Leu Ser Leu Tyr Gly Ala Thr 690 695 700 705 ttggtg caa gac ggc ctg gcc ctg acg gat gtg gtg cct cgg ggc acc 2459 Leu ValGln Asp Gly Leu Ala Leu Thr Asp Val Val Pro Arg Gly Thr 710 715 720 aaggag cat gcc ttc ctg agc gcc cag ctc agg tac ttc tcc ctg tac 2507 Lys GluHis Ala Phe Leu Ser Ala Gln Leu Arg Tyr Phe Ser Leu Tyr 725 730 735 gaggtg gcc ctg gtg acc cag ggt ggc ttt gac tac gcc cac tcc caa 2555 Glu ValAla Leu Val Thr Gln Gly Gly Phe Asp Tyr Ala His Ser Gln 740 745 750 cgcgcc ctc ttt gat ctg cac cag cgc ttc agt tcc ctc aag gcg gtg 2603 Arg AlaLeu Phe Asp Leu His Gln Arg Phe Ser Ser Leu Lys Ala Val 755 760 765 ctgccc cca ccg gcc acc cag gca ccc cgc acc tgg ctg cac tat tac 2651 Leu ProPro Pro Ala Thr Gln Ala Pro Arg Thr Trp Leu His Tyr Tyr 770 775 780 785cgc aac tgg cta cag gga atc cag gct gcc ttt gac cag gac tgg gct 2699 ArgAsn Trp Leu Gln Gly Ile Gln Ala Ala Phe Asp Gln Asp Trp Ala 790 795 800tct ggg cgc atc acc cgc cac tcg tac cgc aat ggc tct gaa gat ggg 2747 SerGly Arg Ile Thr Arg His Ser Tyr Arg Asn Gly Ser Glu Asp Gly 805 810 815gcc ctg gcc tac aag ctg ctc atc cag act gga gac gcc cag gag cct 2795 AlaLeu Ala Tyr Lys Leu Leu Ile Gln Thr Gly Asp Ala Gln Glu Pro 820 825 830ctg gat ttc agc cag ctg acc aca agg aag ctg gtg gac aga gag gga 2843 LeuAsp Phe Ser Gln Leu Thr Thr Arg Lys Leu Val Asp Arg Glu Gly 835 840 845ctg att cca ccc gag ctc ttc tac atg ggg ctg acc gtg tgg gtg agc 2891 LeuIle Pro Pro Glu Leu Phe Tyr Met Gly Leu Thr Val Trp Val Ser 850 855 860865 agt gac ccc ctg ggt ctg gca gcc tca cag gcc aac ttc tac ccc cca 2939Ser Asp Pro Leu Gly Leu Ala Ala Ser Gln Ala Asn Phe Tyr Pro Pro 870 875880 cct cct gaa tgg ctg cac gac aaa tac gac acc acg ggg gag aac ctt 2987Pro Pro Glu Trp Leu His Asp Lys Tyr Asp Thr Thr Gly Glu Asn Leu 885 890895 cgc atc ccg cca gct cag ccc ttg gag ttt gcc cag ttc ccc ttc ctg 3035Arg Ile Pro Pro Ala Gln Pro Leu Glu Phe Ala Gln Phe Pro Phe Leu 900 905910 ctg cgt ggc ctc cag aag act gca gac ttt gtg gag gcc atc gag ggg 3083Leu Arg Gly Leu Gln Lys Thr Ala Asp Phe Val Glu Ala Ile Glu Gly 915 920925 gcc cgg gca gca tgc gca gag gcc ggc cag gct ggg gtg cac gcc tac 3131Ala Arg Ala Ala Cys Ala Glu Ala Gly Gln Ala Gly Val His Ala Tyr 930 935940 945 ccc agc ggc tcc ccc ttc ctc ttc tgg gaa cag tat ctg ggc ctg cgg3179 Pro Ser Gly Ser Pro Phe Leu Phe Trp Glu Gln Tyr Leu Gly Leu Arg 950955 960 cgc tgc ttc ctg ctg gcc gtc tgc atc ctg ctg gtg tgc act ttc ctc3227 Arg Cys Phe Leu Leu Ala Val Cys Ile Leu Leu Val Cys Thr Phe Leu 965970 975 gtc tgt gct ctg ctg ctc ctc aac ccc tgg atg gct ggc ctc ata gtg3275 Val Cys Ala Leu Leu Leu Leu Asn Pro Trp Met Ala Gly Leu Ile Val 980985 990 ctg gtc ctg gcg atg atg aca gtg gaa ctc ttt ggt atc atg ggt ttc3323 Leu Val Leu Ala Met Met Thr Val Glu Leu Phe Gly Ile Met Gly Phe 9951000 1005 ctg ggc atc aag ctg agt gcc atc ccc gtg gtg atc ctt gtg gcctct 3371 Leu Gly Ile Lys Leu Ser Ala Ile Pro Val Val Ile Leu Val Ala Ser1010 1015 1020 1025 gta ggc att ggc gtt gag ttc aca gtc cac gtg gct ctgggc ttc ctg 3419 Val Gly Ile Gly Val Glu Phe Thr Val His Val Ala Leu GlyPhe Leu 1030 1035 1040 acc acc cag ggc agc cgg aac ctg cgg gcc gcc catgcc ctt gag cac 3467 Thr Thr Gln Gly Ser Arg Asn Leu Arg Ala Ala His AlaLeu Glu His 1045 1050 1055 aca ttt gcc ccc gtg acc gat ggg gcc atc tccaca ttg ctg ggt ctg 3515 Thr Phe Ala Pro Val Thr Asp Gly Ala Ile Ser ThrLeu Leu Gly Leu 1060 1065 1070 ctc atg ctt gct ggt tcc cac ttt gac ttcatt gta agg tac ttc ttt 3563 Leu Met Leu Ala Gly Ser His Phe Asp Phe IleVal Arg Tyr Phe Phe 1075 1080 1085 gcg gcg ctg aca gtg ctc acg ctc ctgggc ctc ctc cat gga ctc gtg 3611 Ala Ala Leu Thr Val Leu Thr Leu Leu GlyLeu Leu His Gly Leu Val 1090 1095 1100 1105 ctg ctg cct gtg ctg ctg tccatc ctg ggc ccg ccg cca gag gtg ata 3659 Leu Leu Pro Val Leu Leu Ser IleLeu Gly Pro Pro Pro Glu Val Ile 1110 1115 1120 cag atg tac aag gaa agccca gag atc ctg agt cca cca gct cca cag 3707 Gln Met Tyr Lys Glu Ser ProGlu Ile Leu Ser Pro Pro Ala Pro Gln 1125 1130 1135 gga ggc ggg ctt aggtgg ggg gca tcc tcc tcc ctg ccc cag agc ttt 3755 Gly Gly Gly Leu Arg TrpGly Ala Ser Ser Ser Leu Pro Gln Ser Phe 1140 1145 1150 gcc aga gtg actacc tcc atg acc gtg gcc atc cac cca ccc ccc ctg 3803 Ala Arg Val Thr ThrSer Met Thr Val Ala Ile His Pro Pro Pro Leu 1155 1160 1165 cct ggt gcctac atc cat cca gcc cct gat gag ccc cct tgg tcc cct 3851 Pro Gly Ala TyrIle His Pro Ala Pro Asp Glu Pro Pro Trp Ser Pro 1170 1175 1180 1185 gctgcc act agc tct ggc aac ctc agt tcc agg gga cca ggt cca gcc 3899 Ala AlaThr Ser Ser Gly Asn Leu Ser Ser Arg Gly Pro Gly Pro Ala 1190 1195 1200act ggg tgaaagagca gctgaagcac agagaccatg tgtggggcgt gtggggtcac 3955 ThrGly tgggaagcac tgggtctggt gttagacgca ggatggaccc ctggagggcc ctgctgctgc4015 tgcatccctt ctcccgaccc agctgtcatg ggcctccctg atatccatac agaacagcca4075 ccgatttgca catccaggcc tgtgtgagcc tgtatctgtg tcacttgaga gtgaaagctg4135 gcacttgggg ctgcagtgca gccctgtccc ccttcccacc ccacaccact gcctgcccag4195 ctgaccaagc ctgagggacc ctccagcacc cttccgtctg gtgactcctg ggcaggctct4255 ccatatccct gcccacctcc taccacatcc attatttata tgaaaatgtc tatttttgta4315 gtagacatac atgttagcta tgatgaaagt tttatttttt aaagaatgaa atatattcta4375 tgtgaactct cgtgcc 4391 2 1203 PRT Homo sapiens 2 Met Thr Arg SerPro Pro Leu Arg Glu Leu Pro Pro Ser Tyr Thr Pro 1 5 10 15 Pro Ala ArgThr Ala Ala Pro Gln Ile Leu Ala Gly Ser Leu Lys Ala 20 25 30 Pro Leu TrpLeu Arg Ala Tyr Phe Gln Gly Leu Leu Phe Ser Leu Gly 35 40 45 Cys Gly IleGln Arg His Cys Gly Lys Val Leu Phe Leu Gly Leu Leu 50 55 60 Ala Phe GlyAla Leu Ala Leu Gly Leu Arg Met Ala Ile Ile Glu Thr 65 70 75 80 Asn LeuGlu Gln Leu Trp Val Glu Val Gly Ser Arg Val Ser Gln Glu 85 90 95 Leu HisTyr Thr Lys Glu Lys Leu Gly Glu Glu Ala Ala Tyr Thr Ser 100 105 110 GlnMet Leu Ile Gln Thr Ala Arg Gln Glu Gly Glu Asn Ile Leu Thr 115 120 125Pro Glu Ala Leu Gly Leu His Leu Gln Ala Ala Leu Thr Ala Ser Lys 130 135140 Val Gln Val Ser Leu Tyr Gly Lys Ser Trp Asp Leu Asn Lys Ile Cys 145150 155 160 Tyr Lys Ser Gly Val Pro Leu Ile Glu Asn Gly Met Ile Glu ArgMet 165 170 175 Ile Glu Lys Leu Phe Pro Cys Val Ile Leu Thr Pro Leu AspCys Phe 180 185 190 Trp Glu Gly Ala Lys Leu Gln Gly Gly Ser Ala Tyr LeuPro Gly Arg 195 200 205 Pro Asp Ile Gln Trp Thr Asn Leu Asp Pro Glu GlnLeu Leu Glu Glu 210 215 220 Leu Gly Pro Phe Ala Ser Leu Glu Gly Phe ArgGlu Leu Leu Asp Lys 225 230 235 240 Ala Gln Val Gly Gln Ala Tyr Val GlyArg Pro Cys Leu His Pro Asp 245 250 255 Asp Leu His Cys Pro Pro Ser AlaPro Asn His His Ser Arg Gln Ala 260 265 270 Pro Asn Val Ala His Glu LeuSer Gly Gly Cys His Gly Phe Ser His 275 280 285 Lys Phe Met His Trp GlnGlu Glu Leu Leu Leu Gly Gly Met Ala Arg 290 295 300 Asp Pro Gln Gly GluLeu Leu Arg Ala Glu Ala Leu Gln Ser Thr Phe 305 310 315 320 Leu Leu MetSer Pro Arg Gln Leu Tyr Glu His Phe Arg Gly Asp Tyr 325 330 335 Gln ThrHis Asp Ile Gly Trp Ser Glu Glu Gln Ala Ser Thr Val Leu 340 345 350 GlnAla Trp Gln Arg Arg Phe Val Gln Leu Ala Gln Glu Ala Leu Pro 355 360 365Glu Asn Ala Ser Gln Gln Ile His Ala Phe Ser Ser Thr Thr Leu Asp 370 375380 Asp Ile Leu His Ala Phe Ser Glu Val Ser Ala Ala Arg Val Val Gly 385390 395 400 Gly Tyr Leu Leu Met Leu Ala Tyr Ala Cys Val Thr Met Leu ArgTrp 405 410 415 Asp Cys Ala Gln Ser Gln Gly Ser Val Gly Leu Ala Gly ValLeu Leu 420 425 430 Val Ala Leu Ala Val Ala Ser Gly Leu Gly Leu Cys AlaLeu Leu Gly 435 440 445 Ile Thr Phe Asn Ala Ala Thr Thr Gln Val Leu ProPhe Leu Ala Leu 450 455 460 Gly Ile Gly Val Asp Asp Val Phe Leu Leu AlaHis Ala Phe Thr Glu 465 470 475 480 Ala Leu Pro Gly Thr Pro Leu Gln GluArg Met Gly Glu Cys Leu Gln 485 490 495 Arg Thr Gly Thr Ser Val Val LeuThr Ser Ile Asn Asn Met Ala Ala 500 505 510 Phe Leu Met Ala Ala Leu ValPro Ile Pro Ala Leu Arg Ala Phe Ser 515 520 525 Leu Gln Ala Ala Ile ValVal Gly Cys Thr Phe Val Ala Val Met Leu 530 535 540 Val Phe Pro Ala IlePhe Ser Leu Asp Leu Arg Arg Arg His Cys Gln 545 550 555 560 Arg Leu AspVal Leu Cys Cys Phe Ser Ser Pro Cys Ser Ala Gln Val 565 570 575 Ile GlnIle Leu Pro Gln Glu Leu Gly Asp Gly Thr Val Pro Val Gly 580 585 590 IleAla His Leu Thr Ala Thr Val Gln Ala Phe Thr His Cys Glu Ala 595 600 605Ser Ser Gln His Val Val Thr Ile Leu Pro Pro Gln Ala His Leu Val 610 615620 Pro Pro Pro Ser Asp Pro Leu Gly Ser Glu Leu Phe Ser Pro Gly Gly 625630 635 640 Ser Thr Arg Asp Leu Leu Gly Gln Glu Glu Glu Thr Arg Gln LysAla 645 650 655 Ala Cys Lys Ser Leu Pro Cys Ala Arg Trp Asn Leu Ala HisPhe Ala 660 665 670 Arg Tyr Gln Phe Ala Pro Leu Leu Leu Gln Ser His AlaLys Ala Ile 675 680 685 Val Leu Val Leu Phe Gly Ala Leu Leu Gly Leu SerLeu Tyr Gly Ala 690 695 700 Thr Leu Val Gln Asp Gly Leu Ala Leu Thr AspVal Val Pro Arg Gly 705 710 715 720 Thr Lys Glu His Ala Phe Leu Ser AlaGln Leu Arg Tyr Phe Ser Leu 725 730 735 Tyr Glu Val Ala Leu Val Thr GlnGly Gly Phe Asp Tyr Ala His Ser 740 745 750 Gln Arg Ala Leu Phe Asp LeuHis Gln Arg Phe Ser Ser Leu Lys Ala 755 760 765 Val Leu Pro Pro Pro AlaThr Gln Ala Pro Arg Thr Trp Leu His Tyr 770 775 780 Tyr Arg Asn Trp LeuGln Gly Ile Gln Ala Ala Phe Asp Gln Asp Trp 785 790 795 800 Ala Ser GlyArg Ile Thr Arg His Ser Tyr Arg Asn Gly Ser Glu Asp 805 810 815 Gly AlaLeu Ala Tyr Lys Leu Leu Ile Gln Thr Gly Asp Ala Gln Glu 820 825 830 ProLeu Asp Phe Ser Gln Leu Thr Thr Arg Lys Leu Val Asp Arg Glu 835 840 845Gly Leu Ile Pro Pro Glu Leu Phe Tyr Met Gly Leu Thr Val Trp Val 850 855860 Ser Ser Asp Pro Leu Gly Leu Ala Ala Ser Gln Ala Asn Phe Tyr Pro 865870 875 880 Pro Pro Pro Glu Trp Leu His Asp Lys Tyr Asp Thr Thr Gly GluAsn 885 890 895 Leu Arg Ile Pro Pro Ala Gln Pro Leu Glu Phe Ala Gln PhePro Phe 900 905 910 Leu Leu Arg Gly Leu Gln Lys Thr Ala Asp Phe Val GluAla Ile Glu 915 920 925 Gly Ala Arg Ala Ala Cys Ala Glu Ala Gly Gln AlaGly Val His Ala 930 935 940 Tyr Pro Ser Gly Ser Pro Phe Leu Phe Trp GluGln Tyr Leu Gly Leu 945 950 955 960 Arg Arg Cys Phe Leu Leu Ala Val CysIle Leu Leu Val Cys Thr Phe 965 970 975 Leu Val Cys Ala Leu Leu Leu LeuAsn Pro Trp Met Ala Gly Leu Ile 980 985 990 Val Leu Val Leu Ala Met MetThr Val Glu Leu Phe Gly Ile Met Gly 995 1000 1005 Phe Leu Gly Ile LysLeu Ser Ala Ile Pro Val Val Ile Leu Val Ala 1010 1015 1020 Ser Val GlyIle Gly Val Glu Phe Thr Val His Val Ala Leu Gly Phe 1025 1030 1035 1040Leu Thr Thr Gln Gly Ser Arg Asn Leu Arg Ala Ala His Ala Leu Glu 10451050 1055 His Thr Phe Ala Pro Val Thr Asp Gly Ala Ile Ser Thr Leu LeuGly 1060 1065 1070 Leu Leu Met Leu Ala Gly Ser His Phe Asp Phe Ile ValArg Tyr Phe 1075 1080 1085 Phe Ala Ala Leu Thr Val Leu Thr Leu Leu GlyLeu Leu His Gly Leu 1090 1095 1100 Val Leu Leu Pro Val Leu Leu Ser IleLeu Gly Pro Pro Pro Glu Val 1105 1110 1115 1120 Ile Gln Met Tyr Lys GluSer Pro Glu Ile Leu Ser Pro Pro Ala Pro 1125 1130 1135 Gln Gly Gly GlyLeu Arg Trp Gly Ala Ser Ser Ser Leu Pro Gln Ser 1140 1145 1150 Phe AlaArg Val Thr Thr Ser Met Thr Val Ala Ile His Pro Pro Pro 1155 1160 1165Leu Pro Gly Ala Tyr Ile His Pro Ala Pro Asp Glu Pro Pro Trp Ser 11701175 1180 Pro Ala Ala Thr Ser Ser Gly Asn Leu Ser Ser Arg Gly Pro GlyPro 1185 1190 1195 1200 Ala Thr Gly 3 20 DNA Artificial Sequence Primer3 caaactccaa gggggctctg 20 4 20 DNA Artificial Sequence Primer 4cacaaagccc aagacctgag 20 5 20 DNA Artificial Sequence Primer 5tggaattctt gggtngtngc 20 6 17 DNA Artificial Sequence Primer 6gaytgyttyt gggargg 17

We claim
 1. An isolated and/or recombinantly produced human ptc-2polypeptide.
 2. An isolated and/or recombinantly produced polypeptidecomprising a ptc-2 amino acid sequence which can be encoded by a nucleicacid which hybridizes under stringent conditions to a sequence of SEQID. No.
 1. 3. An isolated and/or recombinantly produced polypeptidecomprising a ptc-2 amino acid sequence which is at least 90% identicalwith the sequence of SEQ ID. No.
 2. 4. An isolated nucleic acidincluding a coding sequence for a human ptc-2 polypeptide.
 5. Anisolated nucleic acid including a coding sequence for a polypeptidecomprising a ptc-2 amino acid sequence, which coding sequence hybridizesunder stringent conditions to a sequence of SEQ ID. No.
 1. 6. A nucleicacid comprising (i) a coding sequence for a polypeptide comprising aptc-2 amino acid sequence, which coding sequence hybridizes understringent conditions to a sequence of SEQ ID. No. 1; and (ii) aheterologous transcriptional regulatory sequence operably linkedthereto.
 7. A cell harboring the nucleic acid of claim 5 or
 6. 8. Anoligonucleotide probe comprising a nucleotide sequence which hybridizesunder stringent conditions to a human ptc-2 gene, but not to a humanptc-1 gene.